Structural Biology of Membrane Proteins RSC

Biomolecular Sciences 1st Edition Reinhard

Grisshammer pdf download

https://ebookname.com/product/structural-biology-of-membrane-
proteins-rsc-biomolecular-sciences-1st-edition-reinhard-
grisshammer/

Get the full ebook with Bonus Features for a Better Reading Experience on ebookname.com
Instant digital products (PDF, ePub, MOBI) available
Download now and explore formats that suit you...

Protein Nucleic Acid Interactions Structural Biology

RSC Biomolecular Sciences 1st Edition Phoebe A. Rice

https://ebookname.com/product/protein-nucleic-acid-interactions-
structural-biology-rsc-biomolecular-sciences-1st-edition-phoebe-
a-rice/

Computational and Structural Approaches to Drug

Discovery Ligand Protein Interactions RSC Biomolecular
Sciences 1st Edition R. Stroud

https://ebookname.com/product/computational-and-structural-
approaches-to-drug-discovery-ligand-protein-interactions-rsc-
biomolecular-sciences-1st-edition-r-stroud/

Membrane Proteins 1st Edition Douglas C. Rees (Eds.)

https://ebookname.com/product/membrane-proteins-1st-edition-
douglas-c-rees-eds/

Hamas and Ideology 1st Edition Shaul Bartal

https://ebookname.com/product/hamas-and-ideology-1st-edition-
shaul-bartal/
Infection Prevention and Control Theory and Practice
for Healthcare Professionals 1st Edition Debbie Weston

https://ebookname.com/product/infection-prevention-and-control-
theory-and-practice-for-healthcare-professionals-1st-edition-
debbie-weston/

Children in Hospitality and Tourism 1st Edition Hugues

Séraphin

https://ebookname.com/product/children-in-hospitality-and-
tourism-1st-edition-hugues-seraphin/

Single Nucleotide Polymorphisms Methods and Protocols

1st Edition Pui-Yan Kwok

https://ebookname.com/product/single-nucleotide-polymorphisms-
methods-and-protocols-1st-edition-pui-yan-kwok/

Understanding Cryptography From Established Symmetric

and Asymmetric Ciphers to Post Quantum Algorithms 2nd
Edition Christof Paar • Jan Pelzl • Tim Güneysu

https://ebookname.com/product/understanding-cryptography-from-
established-symmetric-and-asymmetric-ciphers-to-post-quantum-
algorithms-2nd-edition-christof-paar-jan-pelzl-tim-guneysu/

Eat Drink and Be Wary How Unsafe Is Our Food 1st

Edition Charles M. Duncan

https://ebookname.com/product/eat-drink-and-be-wary-how-unsafe-
is-our-food-1st-edition-charles-m-duncan/
Sample Sizes for Clinical Trials 1st Edition Steven A.
Julious

https://ebookname.com/product/sample-sizes-for-clinical-
trials-1st-edition-steven-a-julious/
Structural Biology of Membrane Proteins
RSC Biomolecular Sciences

Editorial Board

Professor Stephen Neidle (Chairman), The School of Pharmacy, University of London, UK

Dr Simon F. Campbell FRS
Dr Marius Clore, National Institutes of Health, USA
Professor David M J Lilley FRS, University of Dundee, UK

This Series is devoted to coverage of the interface between the chemical and biological sci-
ences, especially structural biology, chemical biology, bio- and chemo-informatics, drug dis-
covery and development, chemical enzymology and biophysical chemistry.

Ideal as reference and state-of-the-art guides at the graduate and post-graduate level.

Titles in the series:

Biophysical and Structural Aspects of Bioenergetics

Edited by Mårten Wikström, University of Helsinki, Finland

Structure-based Drug Discovery: An Overview

Edited by Roderick E. Hubbard, University of York, UK and Vernalis (R&D) Ltd,
Cambridge, UK

Exploiting Chemical Diversity for Drug Discovery

Edited by Paul A. Bartlett, Department of Chemistry, University of California, Berkeley and
Michael Entzeroth, S*Bio Pte Ltd, Singapore

Visit our website on www.rsc.org/biomolecularsciences

For further information please contact:

Sales and Customer Services
Royal Society of Chemistry
Thomas Graham House
Science Park, Milton Road
Cambridge CB4 0WF, UK
Telephone +44 (0)1223 432360, Fax +44 (0)1223 426017, Email [email protected]
Structural Biology of Membrane
Proteins

Edited by

Reinhard Grisshammer and Susan K. Buchanan

Laboratory of Molecular Biology, National Institutes of Health,
Bethesda, Maryland, USA
The cover illustration shows the structure of the outer membrane porin MspA from the soil
bacterium Mycobacter smegmatis. Mycobacteria, which have an unusual outer membrane, are
considered a third group equidistant from the Gram-positive and Gram-negative bacteria. The
MspA protein consists of eight subunits, forming a goblet-like structure around a single cen-
tral channel. The upper part of the goblet consists of eight rim domains. The lower part con-
tains two consecutive β-barrels with 8 × 2 = 16 strands forming the stem and the base regions
of the goblet. For details about crystallization and structure, see the chapter by G.E. Schulz.

ISBN-10: 0-85404-361-6
ISBN-13: 978-0-85404-361-3

A catalogue record for this book is available from the British Library

© The Royal Society of Chemistry 2006

All rights reserved

Apart from fair dealing for the purposes of research for non-commercial purposes or for pri-
vate study, criticism or review, as permitted under the Copyright, Designs and Patents Act
1988 and the Copyright and Related Rights Regulations 2003, this publication may not be
reproduced, stored or transmitted, in any form or by any means, without the prior permission
in writing of The Royal Society of Chemistry, or in the case of reproduction in accordance
with the terms of licences issued by the Copyright Licensing Agency in the UK, or in accor-
dance with the terms of the licences issued by the appropriate Reproduction Rights
Organization outside the UK. Enquiries concerning reproduction outside the terms stated
here should be sent to The Royal Society of Chemistry at the address printed on this page.

Published by The Royal Society of Chemistry,

Thomas Graham House, Science Park, Milton Road,
Cambridge CB4 0WF, UK

Registered Charity Number 207890

For further information see our web site at www.rsc.org

Typeset by Macmillan India Ltd, Bangalore, India

Printed by Henry Ling Ltd, Dorchester, Dorset, UK
Preface
The past few years have seen exciting advances in the field of membrane protein struc-
tural biology. Although membrane proteins still constitute a small fraction of the total
number of solved protein structures (see Hartmut Michel’s and Stephen White’s sum-
maries at http://www.mpibp-frankfurt.mpg.de/michel/public/memprotstruct.html,
http://blanco.biomol.uci.edu/Membrane_Proteins_xtal.html), an exponential increase
of membrane protein structures has been observed1 and is anticipated to continue. This
book addresses a number of issues pertaining to membrane protein structural biology.
The first section describes approaches for expression and purification of membrane
proteins, and a general introduction to detergents. The major challenge in this field is
with eukaryotic membrane proteins, which is reflected by the contributions in this sec-
tion. The second section addresses selected methods for structure determination of
membrane proteins, such as solution and solid-state nuclear magnetic resonance,
atomic force microscopy, electron microscopy including single particle analysis and
lipidic cubic phase crystallization. The final section highlights some of the recently
solved membrane protein structures.
The content of the individual chapters varies from basic introduction to very
detailed description of a particular theme. Therefore, the experienced membrane
protein biochemist, as well as the novice, will find useful information. While we
have endeavored to provide a balanced overview, not all topics could be covered.
Clearly, this book is not an exhaustive reference for all concepts relating to mem-
brane protein structural biology. However, the following paragraphs give some fur-
ther information with key references on topics not addressed by this book.
Heterologous expression and purification of membrane proteins have been cov-
ered by a number of reviews.2–5 Here, the reader will find information on both
prokaryotic and eukaryotic membrane proteins. Until recently, all structures of
eukaryotic membrane proteins, for example the visual pigment rhodopsin,6,7 had
been solved with material from natural sources, reflecting the difficulty of produc-
ing functional eukaryotic membrane proteins in heterologous expression systems.
The new structure of a recombinant mammalian voltage-gated potassium channel,
produced in the yeast Pichia pastoris, is therefore exciting.8
Structure determination of any membrane protein necessitates that the purified
protein retain its native fold and full functionality. Progress on refolding of mem-
brane proteins, deposited as aggregates in the cytoplasm of Escherichia coli, has
been demonstrated for seven-helix G-protein-coupled receptors9,10 and bacterial
outer membrane proteins (for reviews, see Refs. 11 and 12). Likewise, cell-free
in vitro expression has been reported for some membrane proteins,13–15 and the
potential usefulness of this method for generating functional membrane protein for
structural studies is under investigation.
vi Preface
Overproduction of correctly folded membrane proteins is intimately linked to effi-
cient membrane insertion. Great advances have been made in understanding the
mechanism of membrane protein insertion into membranes, but a rational approach
to predicting good ‘overexpressors’ from their amino acid sequences is still lacking.
It is simply fascinating to see how the expression levels of closely related proteins,
such as seven-helix G-protein-coupled receptors, vary in a particular host and under
particular growth conditions (see for example Ref. 16). Two recent topics warrant
highlighting: (a) Elegant work combining theory and biochemical experiments pro-
vides the basic features of how transmembrane helices are recognized by the endo-
plasmic reticulum translocon (‘biological’ hydrophobicity scale17,18). (b) Successful
recombinant overproduction of membrane proteins appears to be linked to avoidance
of stress responses in the host cell19 and can be related to the differential expression
of genes involved in membrane protein secretion and cellular physiology.20 This
implies that simple approaches for increasing recombinant membrane protein yields
may be unsuccessful, emphasizing the benefit of considering a ‘whole-cell’ approach
for developing general strategies to obtain high levels of functional membrane pro-
teins, especially of eukaryotic targets.
The process of membrane protein crystallization usually starts with the extraction of
the target from membranes by using detergents, followed by purification. However, the
yields of purified membrane proteins can be low, which has limited the number of
crystallization parameters to be explored. An obvious (but expensive) solution to this
problem is to conserve protein by using nanoliter-pipetting robots (see for example
Ref. 21). Likewise, smaller crystals can now be analyzed due to improvements in syn-
chrotron beam lines. Another avenue for improving the chance of successful crystal-
lization is to employ Fv or Fab antibody fragments for co-crystallization. This
approach relies on expanding the hydrophilic surface of a membrane protein, facilitat-
ing formation of crystal contacts – the reader is referred to22–26 for more information.
Along the same lines, complexes between an integral membrane protein with little
hydrophilic surface, and its soluble partner, may be more amenable to successful crys-
tallization than the membrane protein alone. The recent structure of the mammalian
voltage-gated potassium channel, Kv1.2-b2, illustrates this concept nicely.8 For the
general principles of protein crystallization, the reader is referred to Refs.27 and 28.
Mass spectrometry has found increasing use for the characterization and analysis
of integral membrane proteins, despite the unfavorable spectrometer response
caused by detergent and lipid molecules associated with membrane proteins. The
reader is referred to the following publications for recent advances in this field.29–33
Another spectroscopic method, utilizing spin labeling for electron paramagnetic
resonance experiments, has provided dynamic structural information on membrane
proteins, in contrast to static views obtained from X-ray crystal structures. This tech-
nique has been applied, for example, to channels,34,35 seven-helix G-protein-coupled
receptors36 and bacterial outer membrane transporters.37
This book presents a selected number of recent membrane protein structures, but
many other exciting structures could not be included here. Among these are ATP-
binding-cassette transporters,38,39 major-facilitator superfamily transporters,40,41 the
sodium/proton antiporter from E. coli,42 a bacterial homolog of a neurotransmitter
transporter,43 bacterial and mammalian potassium channels,8,26,44–46 chloride
Preface vii
channels/transporters,25,47 plant light harvesting complexes48,49 and cytochrome
b6f.50,51 A complete list of all membrane protein structures can be found at the web
sites listed in the first paragraph.
Many integral membrane proteins, including eukaryotic members, can already be
produced in quantities sufficient to consider structural and functional analyses.
Further technological advances will continue to promote the exponential growth of
membrane protein structures. Likewise, protocols to assess the dynamics and func-
tions of membrane proteins continue to be developed, especially in the fields of solu-
tion and solid-state nuclear magnetic resonance. Most importantly, the dedication
and enthusiasm of researchers, combined with good biochemistry, will propel mem-
brane protein structural biology to new levels.

Reinhard Grisshammer and Susan Buchanan

October 2005

Acknowledgment
The research of the editors is supported by the Intramural Research Program of the
NIH, National Institute of Diabetes and Digestive and Kidney Diseases.

References
1. S.H. White, Protein Sci., 2004, 13, 1948–1949.
2. R. Grisshammer and C.G. Tate, Q. Rev. Biophys., 1995, 28, 315–422.
3. C.G. Tate, FEBS Lett., 2001, 504, 94–98.
4. V. Sarramegna, F. Talmont, P. Demange and A. Milon, Cell Mol. Life Sci., 2003,
60, 1529–1546.
5. Special Issue on Overexpression of Integral Membrane Proteins, Biochim.
Biophys. Acta, 2003, 1610, 1–153.
6. K. Palczewski, T. Kumasaka, T. Hori, C.A. Behnke, H. Motoshima, B.A. Fox,
I. Le Trong, D.C. Teller, T. Okada, R.E. Stenkamp, M. Yamamoto, M. Miyano,
Science, 2000, 289, 739–745.
7. J. Li, P.C. Edwards, M. Burghammer, C. Villa and G.F.X. Schertler, J. Mol.
Biol., 2004, 343, 1409–1438.
8. S.B. Long, E.B. Campbell and R. MacKinnon, Science, 2005, 309, 897–903.
9. H. Kiefer, Biochim. Biophys. Acta, 2003, 1610, 57–62.
10. J.L. Baneres, A. Martin, P. Hullot, J.P. Girard, J.C. Rossi and J. Parello, J. Mol.
Biol., 2003, 329, 801–814.
11. S.K. Buchanan, Curr. Opin. Struct. Biol., 1999, 9, 455–461.
12. M. Bannwarth and G.E. Schulz, Biochim. Biophys. Acta, 2003, 1610, 37–45.
13. Y. Elbaz, S. Steiner-Mordoch, T. Danieli and S. Schuldiner, Proc. Natl. Acad.
Sci. USA, 2004, 101, 1519–1524.
14. G. Ishihara, M. Goto, M. Saeki, K. Ito, T. Hori, T. Kigawa, M. Shirouzu and
S. Yokoyama, Protein Expr. Purif., 2005, 41, 27–37.
15. C. Klammt, F. Löhr, B. Schäfer, W. Haase, V. Dötsch, H. Rüterjans, C. Glaubitz
and F. Bernhard, Eur. J. Biochem., 2004, 271, 568–580.
viii Preface
16. M. Akermoun, M. Koglin, D. Zvalova-Iooss, N. Folschweiller, S.J. Dowell and
K.L. Gearing, Protein Expr. Purif., 2005, 44, 65–74.
17. T. Hessa, H. Kim, K. Bihlmaier, C. Lundin, J. Boekel, H. Andersson, I. Nilsson,
S.H. White, G. von Heijne, Nature, 2005, 433, 377–381.
18. T. Hessa, S.H. White and G. von Heijne, Science, 2005, 307, 1427.
19. D.A. Griffith, C. Delipala, J. Leadsham, S.M. Jarvis and D. Oesterhelt, FEBS
Lett., 2003, 553, 45–50.
20. N. Bonander, K. Hedfalk, C. Larsson, P. Mostad, C. Chang, L. Gustafsson and
R.M. Bill, Protein Sci., 2005, 14, 1729–1740.
21. D. Stock, O. Perisic and J. Lowe, Prog. Biophys. Mol. Biol., 2005, 88, 311–327.
22. Y. Zhou, J.H. Morais-Cabral, A. Kaufman and R. MacKinnon, Nature, 2001,
414, 43–48.
23. S. Iwata, C. Ostermeier, B. Ludwig and H. Michel, Nature, 1995, 376, 660–669.
24. C. Hunte, J. Koepke, C. Lange, T. Rossmanith and H. Michel, Structure Fold.
Des., 2000, 8, 669–684.
25. R. Dutzler, E.B. Campbell and R. MacKinnon, Science, 2003, 300, 108–112.
26. Y. Jiang, A. Lee, J. Chen, V. Ruta, M. Cadene, B.T. Chait and R. MacKinnon,
Nature, 2003, 423, 33–41.
27. A. McPherson, Crystallization of Biological Macromolecules, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, NY, 1999.
28. S. Iwata, Methods & Results in Crystallization of Membrane Proteins,
International University Line, La Jolla, California, USA, 2003.
29. M. Cadene and B.T. Chait, Anal. Chem., 2000, 72, 5655–5658.
30. A.B. Weinglass, J.P. Whitelegge, Y. Hu, G.E. Verner, K.F. Faull and H.R.
Kaback, EMBO J., 2003, 22, 1467–1477.
31. A.B. Weinglass, M. Soskine, J.L. Vazquez-Ibar, J.P. Whitelegge, K.F. Faull,
H.R. Kaback and S. Schuldiner, J. Biol. Chem., 2005, 280, 7487–7492.
32. M. Trester-Zedlitz, A. Burlingame, B. Kobilka and M. von Zastrow,
Biochemistry, 2005, 44, 6133–6143.
33. L.L. Ilag, I. Ubarretxena-Belandia, C.G. Tate and C.V. Robinson, J. Am. Chem.
Soc., 2004, 126, 14362–14363.
34. E. Perozo, A. Kloda, D.M. Cortes and B. Martinac, J. Gen. Physiol., 2001, 118,
193–206.
35. L.G. Cuello, D.M. Cortes and E. Perozo, Science, 2004, 306, 491–495.
36. W.L. Hubbell, C. Altenbach, C.M. Hubbell and H.G. Khorana, Adv. Protein
Chem., 2003, 63, 243–290.
37. N. Cadieux, P.G. Phan, D.S. Cafiso and R.J. Kadner, Proc. Natl. Acad. Sci. USA,
2003, 100, 10688–10693.
38. K.P. Locher, A.T. Lee and D.C. Rees, Science, 2002, 296, 1091–1098.
39. C.L. Reyes and G. Chang, Science, 2005, 308, 1028–1031.
40. J. Abramson, I. Smirnova, V. Kasho, G. Verner, H.R. Kaback and S. Iwata,
Science, 2003, 301, 610–615.
41. Y. Huang, M.J. Lemieux, J. Song, M. Auer and D.N. Wang, Science, 2003, 301,
616–620.
42. C. Hunte, E. Screpanti, M. Venturi, A. Rimon, E. Padan and H. Michel, Nature,
2005, 435, 1197–1202.
Preface ix
43. A. Yamashita, S.K. Singh, T. Kawate, Y. Jin and E. Gouaux, Nature, 2005, 437,
215–223.
44. D.A. Doyle, J. Morais-Cabral, R.A. Pfuetzner, A. Kuo, J.M. Gulbis, S.L. Cohen,
B.T. Chait and R. MacKinnon, Science, 1998, 280, 69–77.
45. Y. Jiang, A. Lee, J. Chen, M. Cadene, B.T. Chait and R. MacKinnon, Nature,
2002, 417, 515–522.
46. A. Kuo, J.M. Gulbis, J.F. Antcliff, T. Rahman, E.D. Lowe, J. Zimmer,
J. Cuthbertson, F.M. Ashcroft, T. Ezaki, D.A. Doyle. Science, 2003, 300,
1922–1926.
47. J.A. Mindell, M. Maduke, C. Miller and N. Grigorieff, Nature, 2001, 409,
219–223.
48. Z. Liu, H. Yan, K. Wang, T. Kuang, J. Zhang, L. Gui, X. An and W. Chang,
Nature, 2004, 428, 287–292.
49. R. Standfuss, A.C.T. van Scheltinga, M. Lamborghini and W. Kühlbrandt,
EMBO J., 2005, 24, 919–928.
50. G. Kurisu, H. Zhang, J.L. Smith, and W.A. Cramer, Science, 2003, 302,
1009–1014.
51. D. Stroebel, Y. Choquet, J.L. Popot and D. Picot, Nature, 2003, 426, 413–418.
Contents

Section 1 Expression and Purification of Membrane Proteins

Chapter 1 Refolding of G-Protein-Coupled Receptors 3

Jean-Louis Banères
1 Introduction 3
2 Refolding of Membrane Proteins 4
3 In Vitro Protein Refolding 6
4 GPCR In Vitro Refolding 6
4.1 Resolubilization from Inclusion Bodies 6
4.2 Refolding 7
4.3 Refolding of GPCR Fragments 8
4.4 Refolding of Intact GPCRs 9
5 Conclusion 12
References 13

Chapter 2 Expression of Genes Encoding Eukaryotic

Membrane Proteins in Mammalian Cells 15
Philip J. Reeves
1 Introduction 15
2 Mammalian Cell Hosts and Gene Expression Vectors 16
3 Delivery and Maintenance of Expression Vectors in
Mammalian Cells 16
3.1 Transient Transfection 17
3.2 Stable Transfection 17
3.2.1 A Procedure for Stable Transfection
of HEK293S Cells 18
3.3 Stable Episomal Replication 19
3.4 Viral Infection 19
4 HEK293S Stable Cell Lines for High-Level
Expression of Eukaryotic Membrane Proteins 20
4.1 Constitutive Expression 20
4.2 Tetracycline-Regulated Gene Expression 21
xii Contents
4.3 A Plasmid for Tetracycline-Regulated
Expression of Opsin 22
4.3.1 Construction and Characterization of
HEK293S Stable Cell Lines for
Inducible Gene Expression 23
5 Scale Up of Culture Growth and Rhodopsin
Purification 24
5.1 Growth of HEK293S Cells in Suspension
Culture Using a Bioreactor 24
5.2 Immunoaffinity Purification
of Rhodopsin 25
6 Preparation of Eukaryotic Membrane Proteins
Containing Simple N-Glycans 25
7 Outlook for the Use of HEK293S
Tetracycline-Inducible Cell Lines
for Large-Scale Preparation of
Other Eukaryotic Membrane Proteins 26
References 28

Chapter 3 Expression of Recombinant G-Protein Coupled

Receptors for Structural Biology 29
Filippo Mancia and Wayne A. Hendrickson
1 Introduction 29
1.1 Signal Transduction through G-protein
Coupled Receptors 29
1.2 Structural and Functional Characteristics
of GPCRs 30
1.3 Structure Determination of GPCRs 32
2 Expression of Recombinant GPCRs 34
2.1 Overview 34
2.2 Bacteria as Hosts for the Production of
Functional GPCRs 35
2.3 Production of GPCRs in Stably Transfected
Mammalian Cells 39
2.4 Production of GPCRs via Transient
Transfection or Viral Infection of
Mammalian Cells 42
2.5 Production of GPCRs in Yeast 42
2.6 Production of GPCRs in Insect Cells 43
2.7 “In Vivo” Expression in the Eye 43
2.8 Extra-Membranous Expression Systems 44
Contents xiii
3 Conclusions 44
Acknowledgments 45
References 45

Chapter 4 The Purification of G-Protein Coupled

Receptors for Crystallization 51
Tony Warne and Gebhard F.X. Schertler
1 Introduction 51
1.1 Structural Studies of G-Protein
Coupled Receptors 51
1.2 The Turkey Erythrocyte Beta-Adrenergic
Receptor 52
2 Heterogeneity of Overexpressed Receptors 52
2.1 Heterogeneity of GPCRs due to
Post-Translational Modifications 52
2.1.1 N-Glycosylation 52
2.1.2 Palmitoylation 53
2.1.3 Phosphorylation 54
2.2 Other Sources of Heterogeneity 54
3 Membrane Fractionation, Solubilization,
and Detergent Selection 55
3.1 Detergents for Solubilization 56
3.2 Detergents for Final Purification Steps and
Crystallization 57
4 Purification 60
4.1 Use of Purification Tags and Fusions 60
4.2 Removal of Tags and Fusions 63
4.3 Ligand Affinity Chromatography 63
4.4 Final Purification Steps before Crystallization
and Assembly of Complexes 64
4.5 Lipid Content during Purification 66
5 Final Quality Control, Monitoring Protein Stability,
Aggregational State, Lipid, and Bound Detergent 66
6 Conclusions 68
Acknowledgments 68
References 68

Chapter 5 An Introduction to Detergents and Their

Use in Membrane Protein Studies 72
Fabien Walas, Hiroyoshi Matsumura and Ben Luisi
1 Introduction 72
xiv Contents
2 Physical Properties of Detergents Used in Membrane
Protein Studies 73
2.1 Properties and Classification of
Detergents 74
2.1.1 Properties of Detergents 74
2.1.2 Ionic Detergents 75
2.1.3 Non-Ionic Detergents 76
2.1.4 Zwitterionic Detergents 77
2.1.5 Amphipols 77
2.2 Lipopeptide Detergents 78
2.3 Supplements and Additives for Detergents 78
3 Extraction and Purification Procedure Using
Common Detergents 79
3.1 Choice of Detergent 79
3.2 Purification of Membrane Proteins in the
Presence of Detergents 80
3.2.1 Strategy and Method 80
3.2.2 Detergent Exchange or Removal 81
4 Use of Detergents in Membrane Protein
Crystallization 82
4.1 Introduction 82
4.2 Membrane Protein Crystallization in
Lipid Cubic Phase 83
4.3 Crystal Lattice Organization 83
4.4 Example of Detergent Interactions with β-Sheet
Membrane Proteins 84
4.4.1 Crystal Structure of VceC, an Outer
Membrane Protein from
Vibrio Cholerae 85
4.4.2 Detergent Organization in Crystals of
Monomeric Outer Membrane
Phospholipase A 85
4.5 Example of Detergent and α-Helical Type
Membrane Protein Contact 85
4.5.1 Crystal Structure of Rotor Rings 85
4.5.2 Structure of Bovine Rhodopsin in a
Trigonal Crystal Form 87
4.6 A Synopsis of Detergent–Protein
Interactions in Crystals 87
5 Conclusion 89
Acknowledgements 90
References 90
Contents xv
Section 2 Methods for Structural Characterization of
Membrane Proteins

Chapter 6 Solution NMR Approaches to the

Structure and Dynamics of Integral
Membrane Proteins 99
John H. Bushweller, Tomasz Cierpicki and
Yunpeng Zhou
1 Introduction 99
2 Protein Production and Optimization for
NMR Studies 100
2.1 Protein Production 100
2.2 Sample Optimization 101
3 NMR Methodology for the Study of Integral
Membrane Proteins 102
3.1 High Level Deuteration and Assignment
Strategies Using TROSY-Based Experiments 102
3.2 Carbon Detected Experiments: Breaking
the Limit of Sensitivity 104
3.3 Use of Methyl Protonation to Increase the
Number of Nuclear Overhauser Effect-Derived
Distance Constraints 105
3.4 Application of Electron-Nuclear Relaxation
for Long Range Distances 106
3.5 Residual Dipolar Couplings 106
4 Solution NMR Structures of Helical Integral
Membrane Proteins 107
4.1 F1Fo ATP Synthase Subunit c from E. coli 107
4.2 MerF 108
4.3 Mistic 109
5 Solution NMR Structures of β-Barrel
Membrane Proteins 109
5.1 OmpA 110
5.2 OmpX 110
5.3 PagP 111
6 Solution NMR Characterization of Membrane
Protein Dynamics 111
6.1 OmpA 112
6.2 PagP 112
7 Future Directions 113
References 114
xvi Contents

Chapter 7 Membrane Proteins Studied by

Solid-State NMR 118
Adam Lange and Marc Baldus
1 Introduction 118
2 Sample Preparation and Methodology 118
2.1 Isotope Labelling and Solid-State NMR
Sample Preparation 118
2.2 Resonance Assignments and Structure
Determination 120
3 Applications 121
3.1 Membrane Protein Structure 121
3.2 Ligand Binding to Membrane Proteins 121
3.3 Membrane Protein Dynamics 124
4 Conclusions 125
Acknowledgements 126
References 126

Chapter 8 Assessing Structure and Dynamics of Native

Membrane Proteins 131
W. Kukulski, T. Kaufmann, T. Braun, H. Rémigy,
D. Fotiadis and A. Engel
Abstract 131
1 Introduction 131
2 Assembly of 2D Crystals 132
3 Electron Microscopy 135
3.1 Image Formation 135
3.2 Electron Diffraction 138
3.3 Specimen Preparation 138
3.4 Data Processing 139
4 Atomic Force Microscopy 142
4.1 Image Formation 142
4.2 Sample Preparation 143
4.3 Optimized Imaging Conditions 143
4.4 Imaging Native Membranes 144
4.5 Nanodissection 144
4.6 Image Processing 145
5 Conclusion and Perspectives 147
References 147

Chapter 9 State-of-the-Art Methods in Electron

Microscopy, including Single-Particle Analysis 152
Vinzenz M. Unger
1 Introduction 152
Contents xvii
2 Sample Preparation 153
3 Low-Dose Microscopy 154
3.1 Sample Holders 154
3.2 Radiation Damage and Low-Dose Imaging 157
4 Applications of CryoEM 159
4.1 Single-Particle Approaches 159
4.1.1 Generating a Data Set 160
4.1.2 Particle Classification 160
4.1.3 Euler Angle Determination 163
4.1.4 Structure Calculation 163
4.2 Electron Crystallography 164
4.2.1 2D Crystallization and Advantages
of Crystalline Samples over
Single Particles 165
4.2.2 Image Filtering and Lattice
Straightening 166
4.2.3 Impact of CTF on Images of 2D
crystals and CTF–Correction 167
4.2.4 Projection Density Maps and
Calculation of 3D Structures 168
5 Examples 170
5.1 Single-Particle Reconstructions 170
5.2 Electron Crystallography 170
6 Conclusion 170
References 171

Chapter 10 Atomic Resolution Structures of Integral

Membrane Proteins Using Cubic Lipid Phase
Crystallization 173
Hartmut Luecke
1 Introduction 173
1.1 Nuclear Magnetic Resonance
Techniques 173
1.2 Crystallography 174
1.3 Crystallization Techniques 174
1.3.1 Vapor Diffusion 174
1.3.2 Microdialysis Crystallization 175
1.4 Special Issues of Membrane Protein
Crystallization 175
1.5 History of Membrane Protein
Crystallization 176
1.6 Aim of this Chapter 177
2 Membrane Protein Crystals and Crystallization 177
xviii Contents
2.1 Vapor Diffusion of Detergent-Solubilized
Membrane Proteins 177
2.2 The Cubic Lipid Phase (CLP) Crystallization
Method 179
2.3 Crystallization from Bicelles 185
2.4 Crystallization from Spherical Micelles 186
3 Advantages of Structures in a Native Setting
at High Resolution 186
4 Conclusions 188
References 188

Section 3 New Membrane Protein Structures

Chapter 11 Aquaporins: Integral Membrane

Channel Proteins 195
Robert M. Stroud, William E.C. Harries, John Lee,
Shahram Khademi and David Savage
1 Introduction 195
2 The Exclusion Barrier to Ions and Protons in
Aquaporins 199
2.1 Global Orientational Tuning by the
NPA Motif 199
2.2 Helix Dipole 201
2.3 Electrostatic Desolvation Penalty 201
3 Selectivity in the Aquaporin Family 201
4 Permeation of Substances Other than Water and
Glycerol 203
4.1 Conductance of Other Molecules 203
5 Aquaporin Monomer Associations and
their Functional Implications 204
5.1 The Eye Lens: A Brief History of
Aquaporin 0 Research 204
5.2 Aquaporin 0 Monomer Structure and
Organization 205
5.3 Extracellular Domain Interactions 208
Acknowledgment 209
References 209

Chapter 12 Gas Channels for Ammonia 212

Shahram Khademi and Robert M. Stroud
1 Introduction 212
2 The Structure of Ammonia Channel 213
Contents xix
2.1 Overall Structure of AmtB: A New Family
of 11-Crossing Proteins 213
2.2 A Membrane Protein with In-Plane Quasi
Twofold Symmetry 217
2.3 The Ammonia Pathway 218
3 Reconstituted into Liposomes AmtB Acts
as a Channel that Conducts NH3 222
4 The Mechanism of Conduction 224
4.1 pH-Dependent Effects 224
4.2 Competitive Inhibition 226
4.3 Transmembrane Potential 226
5 The Rh Proteins 227
6 Comparison with Aquaporins 228
7 Comparison with K1 Channel 230
Acknowledgment 230
References 230

Chapter 13 Channels in the Outer Membrane

of Mycobacter 235
Georg E. Schulz
1 Introduction 235
2 Structure Determination 236
2.1 Protein Production 236
2.2 Crystallization 238
2.3 X-Ray Analysis 239
3 Structure Description 240
3.1 The Channel 240
3.2 β-Barrels 242
3.3 Protein Properties 244
4 The Outer Membrane 245
4.1 Membrane Structure 245
4.2 Porin Localization 248
5 Conclusion 249
Acknowledgments 249
References 249

Chapter 14 The Structure of the SecY Protein

Translocation Channel 252
Bert Van Den Berg and Ian Collinson
1 Introduction 252
1.1 The Sec61/SecY Complex 253
1.2 The Three Different Translocation Modes 253
xx Contents
2 Structure Determination of the SecY Complex by
Electron Cryo-Microscopy 255
3 Determination of the X-ray Crystal Structure of the
SecY Complex 257
4 Description of the Structure of the SecY Complex 259
5 Post-Translational Translocation in Bacteria 263
5.1 A Model for Post-Translational Protein
Translocation in Bacteria 265
6 Conclusions and Outlook 265
References 266

Chapter 15 Structure and Function of the Translocator

Domain of Bacterial Autotransporters 270
Peter Van Ulsen, Piet Gros and
Jan Tommassen
1 Introduction 270
2 The NalP Autotransporter 271
3 The Translocator Domain of Autotransporters 272
4 Purification and In Vitro Folding of the NalP
Translocator Domain 273
5 The Structure of the NalP Translocator Domain 277
6 Comparison of the NalP Translocator Domain
to Other Translocator Domains and to TolC 279
7 The Autotransporter Secretion Mechanism 283
References 285

Chapter 16 X-Ray Crystallographic Structures

of Sarcoplasmic Reticulum Ca21-ATPase
at the Atomic Level 288
Jesper Vuust Møller, Poul Nissen and Thomas
Lykke-Møller Sørensen
1 Introduction 288
2 The Transport Scheme and Thermodynamics
of Ca21 Transport 289
3 Overall structure of Ca21-ATPase 290
4 Transport Models 292
5 Initialization of the Cycle: Phosphorylation and
Calcium Ion Occlusion 293
6 The Dephosphorylation Step and Proton Counter
Transport 297
7 Getting Ca21 in and out of the Membrane 300
8 Compact vs. Open Conformations of SERCA 302
Contents xxi
9 Conclusions and Perspectives 303
References 303

Chapter 17 Comparison of the Multidrug Transporter

EmrE Structures Determined by
Electron Cryomicroscopy and X-ray
Crystallography 307
C.G. Tate
1 Introduction 307
2 The Oligomeric State of EmrE 309
3 Transport Activity of EmrE 312
4 Structure of EmrE Determined by Electron
Cryomicroscopy 312
5 Comparison of the EmrE Structure Determined
by Electron Crystallography with a 3.8 Å
Resolution Structure Determined by X-ray
Crystallography 315
6 Conclusions 317
References 317

Chapter 18 Structure of Photosystems I and II 320

Raimund Fromme, Ingo Grotjohann and
Petra Fromme
1 Introduction to Oxygenic Photosynthesis 320
2 Photosystem II 324
2.1 Overview 324
2.2 The Protein subunits in Photosystem II 325
2.2.1 The Core Subunits D1 and D2
(PsbA and PsbD) 325
2.2.2 The Antenna Proteins CP47 and
CP43 (PsbB and PsbC) 325
2.2.3 Cytochrome b559 (PsbE and PsbF) 325
2.2.4 The Small Membrane-Intrinsic
Subunits 327
2.2.5 The Lumenal Subunits PsbO, PsbV,
and PsbU 327
2.3 The Electron Transport Chain of
Photosystem II 327
2.3.1 The Acceptor Site of the Electron
Transport Chain in Photosystem II 328
2.3.2 The Donor Site of the Electron
Transfer Chain of Photosystem II 329
xxii Contents
2.4 The Antenna System of Photosystem II 331
3 Photosystem I 332
3.1 Overview 332
3.2 The Protein Subunits of Photosystem I 332
3.2.1 The Core of Photosystem I: The Large
Subunits PsaA and PsaB 333
3.2.2 The Small Transmembrane Subunits in
Photosystem I 336
3.2.3 The Stromal Hump of PSI: PsaC,
PsaD, and PsaE 338
3.3 The Electron Transfer Chain of
Photosystem I 339
3.3.1 P700: The Primary Electron Donor 340
3.3.2 A: The Initial Electron Acceptor 340
3.3.3 A0: The First Stable Electron Acceptor 341
3.3.4 A1: The Phylloquinone 341
3.3.5 FX: The First FeS Cluster 342
3.3.6 FA and FB: The Terminal FeS Clusters 342
3.4 The Antenna System of Photosystem I 342
3.4.1 The Chlorophylls 343
3.4.2 The Carotenoids 343
3.4.3 The Lipids in Photosystem I 344
4 Conclusion and Outlook 344
References 344

Chapter 19 Glutamate Receptor Ion Channels:

Structural Insights into Molecular
Mechanisms 349
Avinash Gill and Dean R. Madden
1 Introduction 349
1.1 Physiological and Pathophysiological
Roles of the Ionotropic Glutamate Receptors 350
1.2 Medicinal Chemistry 351
1.3 Ionotropic Glutamate Receptor Subunits Are
Modular 351
2 Studies of the Ligand-Binding Domain 351
2.1 Overall Structure 353
2.2 Pharmacological Specificity 353
2.3 Ligand-Binding Domain Conformational
Changes 354
2.4 Correlation with Channel Activation 355
2.5 Dimerization 356
Contents xxiii
2.6 Desensitization and the Stability of the
Dimer Interface 356
3 The Functional Architecture of a Glutamate
Receptor Ion Channel 358
3.1 Structure of a Complete Ionotropic
Glutamate Receptor 358
3.2 The Role of the N-Terminal Domain in
Subunit Assembly 360
3.3 The Organization of the Transmembrane
Domains 361
4 A Working Model of AMPA Receptor Function 362
4.1 LBD Mutations Affecting Binding
and Gating 363
4.2 Subunit Gating Behavior 363
5 Open Questions 364
5.1 Different Models of Partial Agonism 364
5.2 Multistate Kinetic Models 366
5.3 Structural Prospects 366
5.4 Auxiliary Proteins 367
References 367

Chapter 20 The Mitochondrial ADP/ATP Carrier 373

Eva Pebay-Peyroula
1 Introduction 373
2 Mitochondrial Carriers and ADP/ATP Carrier 374
3 Crystallization 375
4 Diffraction, Phasing and Model Building 378
5 Structure Analysis 380
6 Functional Implications 384
6.1 Nucleotide Binding 384
6.2 Conformational Changes 385
6.3 Transport Regulation 386
7 Future Developments and Conclusions 386
Acknowledgments 387
References 387

Subject Index 390

 Section 1

Expression and Purification of

Membrane Proteins
CHAPTER 1

Refolding of G-Protein-Coupled
Receptors
JEAN-LOUIS BANÈRES
UMR 5074 CNRS, “Chimie Biomoléculaire et Interactions Biologiques”, Faculté
de Pharmacie, 15 av. Ch. Flahault, BP14491, 34093 Montpellier Cedex 05, France

1 Introduction
G-protein-coupled receptors (GPCRs) are transmembrane receptors that are involved
in the recognition and transduction of messages as diverse as light, Ca2+ ions, odor-
ants, small molecules (including amino-acids, nucleotides, peptides) and proteins.1,2
Although different classes of receptors have been described,3 they all share a common
structural motif composed of seven α-helices spanning the plasma membrane.
Although significant progress has been made within the last few years in dissect-
ing GPCR-mediated signal transduction pathways, understanding the mechanisms
underlying ligand recognition and signal transduction across the membrane has been
hampered by the lack of information at the molecular level. This is largely due to the
low abundance of most GPCRs in cellular membranes. Furthermore, few expression
systems have proven satisfactory for producing these receptors in a functional state
and sufficient yields.4–6 Structural information on the GPCR family is therefore very
sparse, with the exception of rhodopsin for which X-ray7 and electron8 diffraction
data have been obtained.
Recombinant expression has been one of the major bottlenecks in structural biol-
ogy of GPCRs.9 One of the most widely used expression systems for structural biol-
ogy is Escherichia coli. However, in general, bacterial expression has been
hampered by the relatively low yields of GPCRs owing to the toxic effects caused
by these 7TM receptors when inserted into the bacterial membrane.6 To circumvent
this toxicity problem, GPCRs can be directed to bacterial inclusion bodies. This
leads to high expression levels of the receptor (in the range of 10–50 mg of protein
per liter of bacterial culture). However, the highly expressed recombinant receptors
are inactive and require refolding into a functional form. This explains why intensive
work has been developed during the last years in analyzing the refolding of GPCRs
4 Chapter 1
and in devising efficient strategies for refolding receptors solubilized under denatur-
ing conditions.
Understanding the basic principles of membrane protein folding is also of great
interest in a fundamental perspective. Many studies have been dedicated in the past
years to understand the trafficking of GPCRs.10 The quality control process in the
endoplasmic reticulum involves a variety of mechanisms. These mechanisms ensure
that only correctly folded proteins are directed to the plasma membrane. Despite this
stringent quality control mechanism, gain- or loss-of-function mutations affecting
protein folding in the endoplasmic reticulum, which have been described, can have
profound effects on the health of an organism. Understanding the molecular mecha-
nisms of protein folding could therefore help in correcting the structural abnormali-
ties associated with misfolded receptors.
There are essentially two main types of membrane-spanning structures: trans-
membrane α-helices and β-barrels. The latter appear to be limited to outer mem-
brane proteins. The present work will focus on the refolding of α-helical membrane
proteins. This is the most general case and the most interesting from a pharmaco-
logical point of view. It applies, in particular, to the GPCR family.
Investigating the in vitro refolding of membrane proteins is a difficult task, in par-
ticular, due to the hydrophobic nature of integral membrane proteins. Indeed, to
work with isolated membrane proteins, one has to manipulate refolding solvents,
generally composed of detergents or detergent/lipid mixtures, which poorly mimic
the natural membranes. Nevertheless, biophysical studies on model systems have
begun to provide a sound physical basis for membrane protein folding.11

2 Refolding of Membrane Proteins

As stated above, one of the greatest problems in setting up conditions for studying a
membrane protein in vitro arises from the relative instability of these proteins in deter-
gent solutions. This problem, associated with the low abundance of most GPCRs in
cellular membranes, explains the limited number of examples of refolding studies with
GPCRs. Most of the biophysical studies on α-helical membrane protein folding have
been carried out with bacteriorhodopsin as a model system, since it is a membrane pro-
tein that can be purified in high yields and is relatively stable in solution.12 Moreover,
high-resolution structures of bacteriorhodopsin are available13,14 that help to under-
stand the folding of this protein on a molecular basis. Bacteriorhodopsin functions as
a light-driven proton pump in the purple membrane of the archaebacterium
Halobacterium salinarium.15 As is the case with GPCRs, it possesses seven trans-
membrane α-helical segments. Although it may not be fully representative of GPCRs
at the folding level,16 it is nevertheless a good model system to gain a better under-
standing of receptor folding. Some general rules for α-helical membrane protein fold-
ing have been inferred from folding/unfolding studies with bacteriorhodopsin. In
particular, a two-stage model has been proposed for the folding of these proteins that
decomposes this process.17,18 First, individually stable transmembrane helices form
and then these helices pack to form a functional protein (Figure 1).
The first step in the two-stage model thus involves the formation of the helical
segments, and transmembrane helices display some characteristic features.19 In gen-
eral, they are largely hydrophobic sequences that include a limited number of polar or
Refolding of G-Protein-Coupled Receptors 5

Figure 1 Schematic representation of the two-stage model for membrane protein folding
(18). The transmembrane helices are considered as individually folding units. In the
first stage of the folding, these domains form separately due to specific hydropho-
bic effects and hydrogen bonding. These helical domains then establish intramole-
cular contacts that lead to the native fold

potentially charged groups. In contrast to what is observed in globular proteins, prolines

and glycines are also often found in membrane helices. The bending of a helical seg-
ment induced by a proline residue could be an important feature for membrane protein
function. This is, for example, the case for the GPCR rhodopsin, where kink-inducing
proline residues are found at key positions of the three-dimensional structure.7
Individual transmembrane helical structures are stable essentially because of hydropho-
bic effects and hydrogen bonds that are strong in the low dielectric environment of the
membrane. Many helices found in membrane proteins are likely to be considered as
stable folding units.20 In agreement with this view, individual helical segments or frag-
ments of bacteriorhodopsin or GPCRs, containing only a reduced number of helices,
can reach a stable fold in a membrane mimicking environment (see below).
If individual helices are formed in response to main-chain hydrogen bonding and
hydrophobic effects, other interactions are likely to be involved in their assembly in the
second stage of refolding. An important factor is certainly the way that the helices fit
together, guided by Van der Waals interactions and side-chain rotamers.18,19 Another
factor that is likely to influence the assembly of the transmembrane helices is the lipid
environment. Besides its general role as a solvent, the lipid may also stabilize mem-
brane proteins through specific interactions. Indeed, there are many examples of spe-
cific associations of individual lipids or of classes of lipids with membrane proteins.21–23
The interhelical loops can also have a role in the formation of helix/helix contacts,
although the fact that proteins can in some cases assemble from fragments to form func-
tional species suggests that the constraint induced by the loops may not be essential for
6 Chapter 1
folding. The loops could nevertheless promote folding events that bring transmembrane
helices together. In agreement with this view, in the case of bacteriorhodopsin, the spe-
cific conformation of all the protein loops, except the DE loop, contributes to protein
stability and is required for the correct folding and function of the protein.24
In closing this part, although it may not fully represent the folding process of
GPCRs,16 the two-stage model is certainly a good working basis for a better under-
standing of the folding of membrane proteins. A three stage model has also been pro-
posed that, in addition to the two first steps, introduces a third step corresponding to
ligand binding, folding of extramembranous loops, insertion of peripheral domains
and formation of quaternary structures.25

3 In Vitro Protein Refolding

Inclusion body production is a recurrent theme in recombinant protein technology.
Refolding of inclusion bodies consists first in solubilizing the protein under dena-
turing conditions and then refolding is initiated by the removal of the denaturing
agent. This can be done by dialysis or dilution. Protein folding has also been
achieved by binding the protein to a chromatographic resin in the unfolded state and
subsequent washing with an appropriate buffer that contains no denaturant. Affinity
resins, such as Ni-NTA Sepharose26,27 or heparin Sepharose28 have been used for
poly-histidine-tagged or poly-arginine-tagged proteins, respectively. Compared to
folding by dialysis or rapid dilution, this method has the main advantage of prevent-
ing aggregation due to intermolecular interaction of partly folded protein species.
However, an interference of the chromatographic support with the folding protein
molecule may be detrimental, especially in the case of the highly hydrophobic mem-
brane proteins, possibly causing precipitation of the protein on the matrix.
The efficiency of refolding depends on the competition between protein refolding
and aggregation. One of the main difficulties in refolding membrane proteins is thus
to find conditions that favor refolding over aggregation. A delicate balance must be
reached between too harsh or too mild environments. Protein folding screens for
identification of optimal folding conditions have been developed during the past
years to screen different factors that may influence globular protein refolding.27–32 In
a general manner, this consists in screening multiple conditions, in which different
parameters (additives, pH, salt and protein concentration) are altered. Such folding
screens can be adapted to integral membrane proteins, keeping in mind that one of
the most crucial parameters to test will be the nature of the detergent and/or deter-
gent/lipid mixture. This is indeed likely to be the factor with the most dramatic effect
on membrane protein refolding efficiency.

4 GPCR In Vitro Refolding

4.1 Resolubilization from Inclusion Bodies
In most cases, refolding of GPCRs has been carried out with material recovered from
bacterial inclusion bodies.27,33–35 As far as expression in E. coli is considered, it
seems that there is no general strategy to be used for the efficient accumulation of
Refolding of G-Protein-Coupled Receptors 7
GPCRs in inclusion bodies, even if some rules have been inferred from studies with
different receptors.36 In some cases, the receptor simply fused to a T7 tag was effi-
ciently expressed in E. coli. This is the case, for example, for the leukotriene B4
receptor BLT127 or, more recently, for the V2 vasopressin receptor.37 However, it
must be noted that in the case of BLT1, protein expression levels dramatically vary
from one clone to another. In other cases, fusion of the receptor to a protein partner
was absolutely required for its expression. Different partners, such as glutathione
S-transferase (GST)33,36 or ketosteroid isomerase (KSI)34 have been used. It must be
emphasized again that no system allowing the expression of “all” GPCRs in inclu-
sion bodies has been described so far. For example, no expression was observed with
the 5-HT4a receptor fused to GST, whereas this receptor was efficiently produced
when fused to KSI.34 Similarly, in our hands, among all the receptors we tested, KSI
fusions gave inclusion bodies only with the 5-HT4a receptor. The best approach may
therefore be to test different fusion partners and then quantify the expression levels.
Before starting the refolding, the inclusion body material has to be solubilized.
Globular proteins in inclusion bodies can be solubilized in the presence of high con-
centrations of chaotropic agents, such as guanidinium hydrochloride or urea. In con-
trast, aggregated membrane proteins require detergents (or organic solvents) for
efficient solubilization, due to the predominance of hydrophobic effects in the aggre-
gated material. Usually, SDS is used as a strong denaturing detergent. However, it must
be kept in mind that, in SDS, helical membrane proteins, such as bacteriorhodopsin or
GPCRs are not totally unfolded, but they can retain a significant amount of secondary
structure. Indeed, sequence regions that, in the folded structure, form transmembrane
helices tend to locally adopt an α-helical structure even in SDS. Bacteriorhodopsin, for
example, is about 40–45% helical in SDS.38–40 The 5-HT4a receptor is also 30–35%
helical in SDS-containing buffers (Banères, unpublished data). The SDS-solubilized
starting point for refolding is thus not to be considered as a fully unfolded state but
rather as a partially folded state, as far as the secondary structure is concerned.

4.2 Refolding
As stated above, “unfolded” membrane proteins are first solubilized in harsh deter-
gents, such as SDS or lauroylsarcosine. Refolding will then consist in replacing this
denaturing detergent by a detergent that will stabilize the three-dimensional fold of
the membrane protein. Under these conditions, based on the two-stage model,
regions that have a propensity to fold will do so and then interactions between pro-
tein segments will appear. Those can be intramolecular, leading to refolding or
intermolecular, leading to aggregation. One of the main challenges in refolding
membrane proteins is therefore to find conditions, in particular, the nature of the
detergent environment, which will favor the intramolecular over intermolecular con-
tacts, and therefore refolding over aggregation. This implies finding the right balance
between harsh and mild environments. For GPCRs, however, in the absence of
extensive examples of successful receptor refolding, it is difficult to infer a general
rule for the lipid and detergent requirements for maximal refolding efficiency.
Two different refolding studies have been reported so far for GPCRs. In the first
case, the refolding involved, as described for bacteriorhodopsin, peptides encompassing
8 Chapter 1
one or several transmembrane domains obtained either by peptide synthesis, restricted
protease digestion of receptors or bacterial expression in inclusion bodies.41,42 In the
second case, the intact receptor was produced in bacterial inclusion bodies and then
refolded in vitro.27,34,43

4.3 Refolding of GPCR Fragments

We will not consider here the refolding of the extracellular ectodomains of GPCRs that
has been described for several receptors,29,44,45 since these domains behave as typical
globular proteins. We will focus here only on the transmembrane regions of GPCRs.
Several papers reporting the refolding of fragments containing some of the transmem-
brane helices of GPCRs have been published so far.41,42 These fragments range from a
single transmembrane domain to several transmembrane helices. As predicted by the
two-stage model for membrane protein refolding, these fragments form folded
domains when transferred from denaturing conditions to a milder environment. This
suggests that GPCR transmembrane helices can also be considered as individual fold-
ing units. Besides their interest for a better understanding of the molecular processes
involved in receptor folding, these studies indicate that the study of receptor fragments
could be an alternative to the analysis of the structural properties of GPCRs. We will
provide here two recent examples to illustrate this aspect of GPCR refolding.
The first is that of the µ-opioid receptor for which the refolding of an 80-residue
fragment has recently been described.41 The fragment, produced in E. coli inclusion
bodies as a fusion with GST, encompassed the second and third transmembrane seg-
ments as well as the first extracellular loop of the receptor. In this case, simply by
exchanging the harsh detergent lauroylsarcosine, used for inclusion body solubiliza-
tion, to milder lysophosphatidylcholine micelles, a significant amount of secondary
structure was recovered. Under these conditions, the receptor fragment adopted
approximately 50% α-helical structure, consistent with the assumption of an α-helical
structure in the two membrane-spanning regions and a non-helical structure in the
loop region connecting the second and the third transmembrane domains.
The production of the seven transmembrane domains of the adenosine receptor as
individual synthetic peptides has also been recently reported.42 In this case, circular
dichroism (CD) spectra indicated that each of the seven peptides form stable, inde-
pendent α-helical structures in both detergent micelles and lipid vesicles. In particu-
lar, the peptides corresponding to the third, fourth, sixth and seventh transmembrane
domains exhibit high-helical structure content, close to the predicted maximum for
their transmembrane segments. The peptide corresponding to the first transmembrane
domain also adopts a relatively high content of α-helical structure. Interestingly, the
measured helical content of some transmembrane domains does not directly correlate
with the predicted helicity based on amino acid sequence. This points out that, while
hydrophobic interactions can be a major determinant for folding of transmembrane
peptides, other factors, such as helix–helix interactions may play significant roles for
specific transmembrane domains. Such an observation suggests that, although the
transmembrane peptides may essentially be considered independent folding units,
interactions between the transmembrane domains could be required to some degree
for proper insertion and folding of some helical domains.
Refolding of G-Protein-Coupled Receptors 9
4.4 Refolding of Intact GPCRs
In vitro refolding of some intact receptors has also been reported during the last 10
years. The first bacterially expressed GPCR to be successfully refolded in vitro has
been the OR5 olfactory receptor.43 Subsequently, the refolding of two other GPCRs,
namely the leukotriene B4 receptor BLT127 and the serotonin receptor 5-HT4a,34 has
been described. For all three receptors, the same general approach was used (see
Table 1), and the reader is referred to Ref. 27, 34 and 43 for a step-by-step procedure.
They all were produced in E. coli IB, thus allowing large protein quantities to be
recovered (in the 10 mg range). The receptors were then solubilized with a harsh
detergent. In the case of OR5, the overexpressed protein was solubilized in the strong,
negatively charged detergent, lauroylsarcosine, whereas BLT1 and the 5-HT4a recep-
tors were solubilized in the presence of both urea and SDS. The receptors were then
refolded by solvent exchange. In all cases, solvent exchange was carried out with the
receptor immobilized on a solid Ni-NTA matrix. This could indicate that for integral
membrane proteins, preventing non-specific protein/protein interactions that favor
aggregation, could be a crucial point to get acceptable refolding yields. At least in our
hands, the matrix-assisted procedure systematically yielded significantly higher
refolding yields compared to dialysis or dilution (Banères, unpublished data).
Lacking a detailed theoretical understanding of the various factors affecting
GPCR refolding yields and tendency to aggregate, the strategy used in all the
reported cases of receptor refolding was to vary different parameters in a systematic
way and quantify the refolding yield. One of the most crucial parameters was, as
expected, the composition of detergent/lipid micelles. The OR5 was first reconsti-
tuted in the non-denaturing detergent digitonin. In this detergent, the receptor was
able to bind its ligand (fluorescence-monitored ligand-binding assays) indicating

Table 1 Summary of the expression, solubilization and refolding conditions for

three efficiently refolded GPCRs, the OR5 receptor,43 the BLT1 receptor27
and the 5-HT4a receptor.34 CHS: cholesteryl hemisuccinate, CHAPS:
3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate, DDM:
dodecyl maltoside, DMPC: dimyristoyl-phosphatidylcholine, GST: glu-
tathione S-transferase, KSI: ketosteroid isomerase, IB: E. coli inclusion
bodies, LDAO: lauryl-N,N-dimethylamine-N-oxide, POPC: 1-palmitoyl-2-
oleoyl-sn-glycerol-3-phosphocholine and POPG: 1-palmitoyl-2-oleoyl-sn-
glycero-3-[phospho-rac-(1-glycerol)]. The lipid and/or lipid/detergent
weight ratios used for refolding are indicated
Receptor Expression Solubilization Refolding Detergent References
method and/or lipid
OR5 as IB sarcosyl matrix-assisted POPC/POPG 43
GST fusion (Ni-NTA) (4:1)
BLT1 as IB urea/SDS matrix-assisted LDAO 27
T7-tag fusion (Ni-NTA)
5-HT4a as IB urea/SDS matrix-assisted CHAPS/DMPC/CHS 34
KSI fusion (Ni-NTA) (2:1:0.02)
10 Chapter 1
that, under these conditions, the receptor was properly folded. Subsequently, mixed
micelles, composed of dodecyl maltoside (DDM) as the detergent, and of 1-palmitoyl-
2-oleoyl-sn-glycerol-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-
glycero-3-[phospho-rac-(1-glycerol)] (POPG) as the lipids, were added and the
detergent was removed by treatment with hydrophobic beads. Under these conditions,
the protein was stabilized into a fully active state as assessed by photoaffinity label-
ing. In the case of the BLT1 receptor, the best refolding yields were achieved with
lauryl-N,N-dimethylamine-N-oxide (LDAO) as a detergent. For BLT1, different
detergents were tested and the best yields were systematically achieved from those
with a long alkyl chain, i.e. above C12. The observation of a prominent role of the
alkyl chain length on BLT1 refolding is reminiscent of what had been previously
observed with rhodopsin, where detergents with long alkyl chains (above C10) stabi-
lized the protein better than detergents with shorter chains.46 It is to be noted that
BLT1 and OR5 (see above), were first refolded in solutions containing only deter-
gents. However, in this case, subsequent reconstitution of the receptor in a deter-
gent/lipid mixed micelle or in a lipid vesicle dramatically increases the stability of the
isolated BLT1 receptor in solution (Banères, unpublished data). This suggests that,
even if the detergent can mimic the membrane environment for receptor refolding, the
presence of lipids and maybe specific lipid/protein contacts, are essential to ensure a
full stabilization of the protein in solution. Finally, the 5-HT4a receptor was stabilized
in a functional conformation only in the presence of mixed detergent/lipid micelles. The
micelles in this case were composed of 3-[(3-cholamidopropyl)dimethylammonio]-
1-propanesulfonate (CHAPS), dimyristoyl-phosphatidylcholine (DMPC) and cho-
lesterol (CH) (see Table 1). In contrast to what had been observed with BLT1, the
5-HT4a receptor could not be refolded in the presence of a detergent only, empha-
sizing the importance of lipids for the stabilization of the receptor conformation.
Also, the presence of CH was required not only for increasing the refolding yields
of the 5-HT4a receptor, but also to increase the long-term stability and optimize the
ligand-binding properties of the receptor in solution (see Figure 2). The differences
in the detergent requirements for stabilizing the functional conformations of the
OR5, BLT1 and 5-HT4a receptors indicate that each protein may be a specific case
in terms of detergent and lipid requirements for refolding. In the absence of more
published data, it is difficult to infer a general rule as to which are the most appro-
priate detergent and lipids for GPCR refolding. The most straightforward strategy to
find suitable folding conditions seems therefore to test different detergents and deter-
gent/lipid mixtures in a systematic way.
Besides the step involving the search for a detergent that could promote receptor
folding, the data obtained for the receptors cited above emphasize the fact that one
of the main problems, when screening for the best conditions for protein folding, is
to determine the yield of functional protein. This is easier to achieve for membrane
proteins that display well-defined ligand-binding properties. In the case of OR5,
BLT1 and 5-HT4a, the main criterion used to assess the correct refolding was the lig-
and-binding properties of the reconstituted receptor. It is to be noted in this context
that one must be cautious while using an agonist to determine if the receptor has
been successfully refolded. In the case of an isolated receptor, the agonist affinity
may be significantly lower in the absence of G-proteins. Receptors isolated from
Refolding of G-Protein-Coupled Receptors 11

Figure 2 Percentage of detergent-soluble and functional (i.e. competent to bind antagonist)

5-HT4a receptor after refolding in different detergent and/or detergent/lipid
mixtures. (1) octyl glucoside, (2) LDAO, (3) CHAPS, (4) CHAPS/DMPC (detergent/
lipid ratio 2:1), (5) CHAPS/DOPC (2:1), (6) CHAPS/DMPC/CHS (2:1:0.02), and
(7) CHAPS/DOPC/CHS (2:1:0.02) (see legend to Table 1 for the abbreviations of the
detergents and lipids)

membrane fractions may still be bound to endogenous G-proteins, and hence display
higher agonist affinities. Other parameters can also be used to assess correct folding
of isolated GPCRs. These include the ability of the refolded receptor to interact with
and/or to activate intracellular partners, such as G-proteins or arrestins.47,48
Determining the amount of correctly refolded protein is more difficult when no bio-
logical assay is available. Centrifugation or filtration can remove precipitates result-
ing from protein aggregation, but solubility cannot be used as a stringent criterion
simply because partially folded non-active intermediates can be soluble in the pres-
ence of detergents. Another method that has been largely applied to the refolding of
globular proteins is limited proteolysis. Partially folded intermediates are assumed
to be more susceptible to limited proteolysis than the fully folded protein. However,
despite this method having successfully been applied to globular proteins,31 one
must be cautious when working with membrane proteins, since inaccessibility to the
protease could simply be due to the masking of the cleavage sites by the detergent
and not to the correct folding of the protein. This method has nevertheless been suc-
cessfully applied to membrane proteins, but rather to test, for example, for correct
insertion in a lipid membrane.49,50 Spectroscopic methods, such as CD or fluores-
cence, that give access to the structural characteristics of the protein, can also be
used to monitor refolding. CD will provide the secondary structure content of the
protein,51 whereas tryptophan fluorescence spectroscopy can be used to detect
folded protein, since unfolded conformations, folding intermediates and fully folded
proteins may be distinguishable in their respective spectra.52 However, one must
again be cautious while using these criteria to assess for correct membrane protein
folding. For example, a good CD profile does not mean that the protein is well
folded. If one considers the two-stage model for protein refolding, a possibility is to
get a partially folded protein where the secondary structure elements are formed so
that its far-UV CD properties are close to those of the fully folded protein, but where
the intramolecular helical contacts are not properly established so that the receptor
12 Chapter 1
is not able to bind its ligands. One must also keep in mind that even in harsh deter-
gents, such as SDS, helical membrane proteins, bacteriorhodopsin or GPCRs usually
can retain a certain amount of secondary structure. A good alternative in this case
could be the use of CD in the near-UV regions, rather than in the far-UV regions.
Indeed, the near-UV region is sensitive to the three-dimensional folding of the pro-
tein and is therefore likely to be affected by the packing of the transmembrane
helices.27,34 As for fluorescence, it can be difficult to assess correct refolding in the
absence of a reference spectrum of the functional protein. In a general manner, for
most of the methods described above, one of the main problems indeed arises from
the absence of well-folded protein to be taken as a reference due to the low abun-
dance of most GPCRs in cellular membranes. Finally, as a consequence of the pos-
sible occurrence of detergent-soluble misfolded proteins, a proper way to purify the
functional receptor is also crucial. In this context, the best method seems to be,
whenever possible, affinity chromatography with an immobilized ligand column,
since the main goal of this purification step is to discriminate between active and
inactive receptors.
To give an example emphasizing the importance of a functional assay for assess-
ing receptor refolding, we can consider the case of the 5-HT4a receptor. When
refolding was carried out with only detergents, two different protein fractions were
recovered after the refolding step. The first one, highly aggregated, was simply
removed by centrifugation on a sucrose gradient. The second one was totally solu-
ble in the detergent-containing solution and its secondary structure properly recov-
ered as assessed by far-UV CD. However, only a small amount of receptor in this
fraction was able to bind a 5-HT4a antagonist ligand, as assessed by direct ligand-
binding experiments (Figure 2). It is only after adding lipids in the refolding buffers
that a fully functional protein fraction was recovered.

5 Conclusion
Membrane protein refolding, in particular GPCR refolding, has been the focus of
intensive work during the past years. This is due to the possible implications of
receptor misfolding in some diseases as well as to the interest of GPCR refolding in
the context of high-yield protein production for structural studies. Indeed, the
increasing number of reports of production of GPCRs in E. coli inclusion bodies
makes refolding a central step for producing functional receptors. Although further
development of the techniques and refolding systems are still required, an under-
standing of the process is being gained. The possibility to study the refolding of
model proteins, such as bacteriorhodopsin, and now a few GPCRs, will certainly
provide us with a more detailed model of the refolding mechanism. It is likely that
future success in refolding more membrane proteins and/or reaching higher refold-
ing yields in vitro will depend on how we understand the structural properties of the
membrane proteins, as well as the way they interact with their lipid environment. In
particular, some work will certainly have to focus on the factors that influence the
interactions between helical domains in the membrane protein since this is, at least
in our hands, the limiting step for reaching efficient refolding of all the GPCRs we
have studied so far. Such fundamental work on receptor refolding will without doubt
Refolding of G-Protein-Coupled Receptors 13
help in finding the factors that currently limit high-level in vitro refolding of GPCRs,
especially since high-level expression of “unfolded proteins” seems now somehow
to be ensured, at least in the bacterial system.

References
1. J. Bockaert and J.-P. Pin, EMBO J., 1999, 18, 1723.
2. J. Bockaert, S. Claeysen, C. Becamel, S. Pinloche and A. Dumuis, Int Rev.
Cytol., 2002, 212, 63.
3. S.M. Foord, T.I. Bonner, R.R. Neubig, E.M. Rosser, J.P. Pin, A.P. Davenport,
M. Spedding and A.J. Harmar, Pharmacol. Rev., 2005, 57, 279–288.
4. C.G. Tate and R. Grisshammer, Trends Biotechnol., 1996, 14, 426.
5. C.G. Tate, FEBS Lett., 2001, 504, 94.
6. V. Sarramegna, F. Talmont, P. Demange and A. Milon, Cell. Mol. Life Sci., 2003,
60, 1529.
7. K. Palczewski, T. Kumusaka, T. Hori, C.A. Behnke, H. Motoshima, B.A. Fox,
I. Le Trong, D.C. Teller, T. Okada, R.E. Stenkamp, M. Yamamoto and
M. Miyamo, Science, 2000, 289, 739.
8. J.J. Ruprecht, T. Mielke, R. Vogel, C. Villa and G.F. Schertler, EMBO J., 2004,
23, 3609.
9. K. Lundstrom, Trends Biotechnol., 2005, 23, 103.
10. C.M. Tan, A.E. Brady, H.H. Nickols, Q. Wang and L.E. Limbird, Annu. Rev.
Pharmacol. Toxicol., 2004, 44, 559.
11. P.J. Booth and A.R. Curran, Curr. Opin. Struct. Biol., 1999, 9, 115.
12. J.K. Lanyi and H. Luecke, Curr. Opin. Struct. Biol., 2001, 11, 415.
13. K. Edman, P. Nollert, A. Royant, H. Belrhali, E. Pebay-Peyroula, J. Hajdu,
R. Neutze and E.M. Landau, 1999, Nature, 401, 822.
14. H. Luecke, B. Schobert, H.T. Richter, J.P. Cartailler and J.K. Lanyi, J. Mol.
Biol., 1999, 291, 899.
15. J.K. Lanyi, Annu. Rev. Physiol., 2004, 66, 665.
16. J. Klein-Seetharaman, Trends Pharmacol. Sci., 2005, 26, 183.
17. J.-L. Popot, S.E. Gerchman and D.M. Engelman, J. Mol. Biol., 1987, 198, 655.
18. J.-L. Popot and D.M. Engelman, Biochemistry, 1990, 29, 4031.
19. J.-L. Popot and D.M. Engelman, Annu. Rev. Biochem., 2000, 69, 881.
20. J.-L. Popot, Curr. Opin. Struct. Biol., 1993, 3, 532.
22. A.D. Ferguson, E. Hofmann, J.W. Coulton, K. Diederichs and W. Welte,
Science, 1998, 282, 2215.
23. H. Belrhali, P. Nollert, A. Royant, C. Menzel, J.P. Rosenbuch, E.M. Landau and
E. Pebay-Peyroula, Struct. Fold Des., 1999, 7, 909.
24. J.-M. Kim, P.J. Booth, S.J. Allen and H.G. Khorana, J. Mol. Biol., 2001, 308,
409.
25. D.M. Engelman, Y. Chen, C.N. Chin, A.R. Curran, A.M. Dixon, A.D. Dupuy,
A.S. Lee, U. Lehnert, E.E. Matthews, Y.K. Reshetnyak, A. Senes and J.-L.
Popot, FEBS Lett., 2003, 555, 122–125.
25. M. Weik, G. Zaccaï, N.A. Dencher, D. Oesterhelt and T. Hauss, J. Mol. Biol.,
1998, 275, 625.
14 Chapter 1
26. J.-L. Banères, F. Roquet, M. Green, H. LeCalvez and J. Parello, J. Biol. Chem.,
1998, 273, 24744.
27. J.-L. Banères, A. Martin, P. Hullot, J.-P. Girard, J.-C. Rossi and J. Parello,
J. Mol. Biol., 2003, 329, 801.
28. G. Stempfer, B. Hall-Neugebauer and R. Rudolph, Nat. Biotechnol., 1996, 14,
329.
29. G.Q. Chen and E. Gouaux, Proc. Natl. Acad. Sci. USA, 1997, 94, 13431.
30. N. Armstrong, A. de Lencastre and E. Gouaux, Protein Sci., 1999, 8, 1475.
31. C. Heiring and Y.A. Muller, Protein Eng., 2001, 14, 183.
32. D.A. Tobbell, B.J. Middleton, S. Raines, M.R. Needham, I.W. Taylor, J.Y.
Beveridge and W.M. Abbott, Protein Exp. Purif., 2002, 24, 242.
33. H. Kiefer, K. Maier and R. Vogel, Biochem. Soc. Trans., 27, 908.
34. J.-L. Banères, D. Mesnier, A. Martin, L. Joubert, A. Dumuis and J. Bockaert,
J. Biol. Chem., 2005, 280, 20253.
35. H. Kiefer, Biochim. Biophys. Acta, 2003, 1610, 57.
36. H. Kiefer, R. Vogel and K. Maier, Receptor. Channel., 2000, 7, 109.
37. C. Tian, R.M. Breyer, H.J. Kim, M.D. Karra, D.B. Friedman, A. Karpay and
C.R. Sanders, J. Am. Chem. Soc., 2005, 127, 8010.
38. K.S. Huang, H. Bayley, M.J. Liao, E. London and H.G. Khorana, J. Biol. Chem.,
1981, 256, 3802.
39. E. London and H.G. Khorana, J. Biol. Chem., 1982, 257, 7003.
40. P.J. Booth, Fold. Des., 1997, 2, R85.
41. A. Kerman and V.S. Ananthanarayanan, Biochim. Biophys. Acta, 2005, 1747,
133.
42. T. Lazarova, K.A. Brewin, K. Stoeber and C.R. Robinson, Biochemistry, 2004,
43, 12945.
43. H. Kiefer, J. Krieger, J.D. Olszewski, G. Von Heijne, G.D. Prestwich and
H. Breer, Biochemistry, 1996, 35, 16077.
44. U. Grauschopf, H. Lilie, K. Honold, M. Wozny, D. Reusch, A. Esswein,
W. Schafer, K.P. Rucknagel and R. Rudolph, Biochemistry, 2000, 39, 8878.
45. H. Vlase, N. Matsuoka, P.N. Graves, R.P. Magnusson and T.F. Davies,
Endocrinology, 1997, 8, 1658.
46. P. Knudsen and W.L. Hubbel, Membr. Biochem., 1978, 1, 297.
47. B. Bertin, M. Freissmuth, R.M. Breyer, W. Schutz, A.D. Strosberg and
S. Marullo, J. Biol. Chem., 1992, 267, 8200.
48. T.A. Key, T.A. Bennett, T.D. Foutz, V.V. Gurevich, L.A. Sklar and E.R.
Prossnitz, J. Biol. Chem., 2001, 276, 49204.
49. J.-L. Rigaud, A. Bluzat and S. Büschlen, Biochem. Biophys. Res. Comm., 1983,
111, 373.
50. J. Cladera, J.-L. Rigaud and M. Dunach, Eur. J. Biochem., 1997, 243, 798.
51. G.D. Fasman, Circular Dichroism and the Conformational Analysis of
Biomolecules, Plenum Press, New York, 1996.
52. C.A. Royer, Methods Mol. Biol., 1995, 40, 65.
CHAPTER 2

Expression of Genes Encoding

Eukaryotic Membrane Proteins
in Mammalian Cells
PHILIP J. REEVES
Department of Biological Sciences, University of Essex, Wivenhoe Park,
Colchester CO4 3SQ, UK

1 Introduction
During the last decade there have been significant technological advances in the
development of procedures for functional expression of membrane proteins in mam-
malian cells. Membrane proteins are considerably more difficult to produce and
purify than soluble proteins and this is reflected by the limited number of membrane-
protein structures that have been solved. Obtaining large quantities of higher eukary-
otic membrane proteins in functional form is further complicated due to our limited
knowledge on their folding requirements. Eukaryotic membrane proteins often
exhibit elaborate co/post-translational modifications including N-acetylation,
N- and/or O-linked glycosylation, disulphide bond formation and fatty acylation.
Mammalian cells are capable of carrying out these modifications and are also likely
to have the appropriate cellular chaperones to assist in membrane-protein folding.
Furthermore, the membrane phospholipid composition of mammalian cells is most
likely to provide a suitable environment for promoting correct membrane-protein
insertion and ensuring membrane-protein stability.
For preparation of eukaryotic membrane proteins it would seem reasonable there-
fore to first attempt production in a higher eukaryotic host. However, the perceived
technical difficulties and high costs associated with mammalian cell culture deter
many researchers and instead conventional expression systems, such as the “user-
friendly” prokaryote Escherichia coli, or lower eukaryotes such as yeast are often tried
first. E. coli is considered a workhorse for production of diverse proteins and benefits
from decades of investment in development of bacterial genetic tools and fermentation
technology. Although examples are limited, E. coli, has been used successfully for
Exploring the Variety of Random
Documents with Different Content
 172 THE TOILERS OF THE SEA. rubbed up, as it were. They
would have been like iyory had it not been for some greenish
patches of seaweed. The cartilaginous partitions were delicately
smoothed down and spared. The tomb sometimes produces such
sinister jeweller's work as this. The corpse was interred, as it were,
beneath the dead crabs ; Gilliatt disinterred it. All at once he bent
over hastily. He had just caught sight of a sort of band around the
vertebral column. It was a leather belt which had evidently been
buckled round the man's abdomen when he had been alive. The
leather was mildewed. The buckle was rusted. Gilliatt drew the belt
towards him. The vertebrae resisted, and he was obliged to break
them in order to obtain it. The belt was intact. A crust of limpets had
begun to form upon it. He fingered it and felt a hard object, square
in form, in the interior. Such a thing as undoing the buckle was not
to be thought of. He split the leather with his knife. The belt
contained a small iron box, and a few gold pieces. Gilliatt counted
twenty guineas. The iron box was a sailor's old tobacco box, which
opened with a spring. It was much rusted and very securely closed.
The spring, completely oxidyzed, no longer worked. His knife once
more extricated Gilliatt from his dilemma. A thrust from the point of
the blade caused the cover of the box to fly off. The box opened.
There was nothing in it but paper. A little package of very thin sheets
folded in four covered the bottom of the box. They were damp but
not spoiled. The box, hermetically sealed, had preserved them.
Gilliatt unfolded them. They were three bank notes for a thousand
pounds sterling each, making altogether, seventy-five thousand
francs.
 NOTHING IS HIDDEN AND NOTHING IS LOST. 173 Gilliatt
folded them up again, replaced them in the box, took advantage of
the small space which remained to add the twenty guineas, and
closed the box again, as well as he could. Then he began an
examination .of the belt. The leather, originally glazed on the
outside, was rough on the interior. On that unfinished background
some letters were traced in thick ink. Gilliatt deciphered these
letters: « Sieur Clubin."
 CHAPTER V. IN THB INTERVAL WHICH SEPARATES SIX
INCHES FROM TWO FEET, THERE IS ROOM TO LODGE DEATH.
GiLLiATT replaced the box in the belt and put the belt in the pocket
of his trousers. He left the skeleton to the crabs, with the dead
octopus beside it. While Gilliatt had been engaged with the octopus
and the skeleton, the rising tide had flooded the entrance to the
passage. Gilliatt could get out only by diving under the arch. He
managed it without difficulty ; he knew the exit and he was a master
of these gymnastics of the sea. The reader has had a view of the
drama which had taken place six weeks before. One monster had
seized another. The octopus had caught Clubin. This had been, in
the inexorable gloom, what might almost be called the encounter of
hypocrisies. At the bottom of the abyss, these two existences, made
up of waiting and of shadows, had come into violent collision, and
one, which was the beast, had executed the other, which was the
soul. Sinister justice. The crab feeds on carrion, the octopus feeds
on crabs. The octopus arrests in its passage any swimming animal, a
dog or a man, if it can, drinks liis blood and leaves the dead body at
the bottom of the water. Crabs are the necrophagus beetles of the
sea. Decaying flesh attracts them ; they come ; they eat the corpse ;
the octopus eats them. Dead things disappear in the crab, the crab
disappears in the octopus. We have already pointed out this law. 174
 ItOOM TO LODGE DEATH. 175 Clubin had served the
octopus as bait. The octopus had held him down and drowned him ;
the crabs had devoured him. Some wave had thrust him into the
cave, at the bottom of whose niche Gilliatt had found him. Gilliatt
retraced his steps, fumbling among the rocks in search of sea
urchins and whelks, as he no longer desired any crabs. It would
have seemed to him as though he were devouring human flesh.
Moreover, his only thought was to sup as well as possible before
setting out. Henceforth, nothing detained him. Great tempests are
always followed by a calm, which sometimes lasts many days. No
danger now existed on the side of the sea. Gilliatt was resolved to
set out on the morrow. It was important to keep the barricade
between the Douvres in place during the night, because of the tide ;
but Gilliatt intended to remove this barrier at daybreak, to push the
boat clear of the Douvres, and to set sail for Saint-Sampson. The
light breeze which was blowing from the southwest was precisely the
wind which he needed. The first quarter of the May moon was
beginning ; the days were long. When Gilliatt, having finished his
prowling among the rocks and almost satisfied his appetite, returned
to the gap between the Douvres where his boat lay, the sun had set,
the twilight was livid with that half moonlight which may be called
the light of the crescent ; the tide had attained its height, and was
beginning to ebb again : the smoke stack of the engine, rising
upright above the paunch, had been covered by the foam of the
tempest with a layer of salt, which shone white in the moonlight.
This reminded Gilliatt that the squall had thrown a great deal of rain
water and sea water into his boat, and that, if he wished to set out
on the morrow, he must bale out the paunch. He had noticed, when
he quitted the boat to go in pur 
 176 TSE TOILERS OF THE SEA. suit of crabs, that there
were about six inches of water in the hold. His baling scoop would
be sufficient to throw out this water. On reaching the boat, Gilliatt
gave a start of terror. There were nearly two feet of water in the
boat. A formidable fact, the boat was leaking. It had gradually filled
during Gilliatt's absence. Loaded as it was, twenty inches of water
was a dangerous addition. A little more and it would sink. If Gilliatt
had returned one hour later, he would probably have found only the
mast and smoke stack out of water. Not a moment could be spared
for deliberation. He must find the leak, stop it up, then empty the
boat, or at least relieve it. The pumps of the Durande had been lost
in the shipwreck; Gilliatt was reduced to the baling scoop of the
paunch. He must find the leak, first of all. That was the most urgent
point. Gilliatt set to work instantly, without even giving himself time
to dress again, all shivering as he was. He was no longer conscious
of either hunger or cold. The boat continued to fill. Fortunately,
there was no wind. The slightest swell would have sunk it. The moon
set. Gilliatt searched for a long time, groping, bent, half submerged
in the water. At last he discovered the injury. During the storm, at
the critical moment when the paunch had twisted round, the strong
bark had come somewhat violently in contact with the rocks. One of
the projections of the little Douvre had made a fracture in the hull on
the starboard side. This leak was vexatiously, one might say
treacherously, situated near the joints of two riders, and this,
together with the confusion of the storm, had prevented Gilliatt's
perceiving the injury, in his obscure and rapid survey at the height of
the tempest.
 ROOM TO LODGE DEATH. 177 The fracture had this
alarming feature, tliat it was large, and this reassuring feature that,
although submerged for the moment, by the inward increase of
water, it was above the water line. At the instant when the crack had
opened, the waves had been rudely shaken in the strait, and there
was no longer any smooth water, the water had dashed through the
breach into the boat, the boat beneath this extra load had settled
several inches, and even after the waves had subsided, the weight
of the water which had filtered in, by raising the water line, had kept
the leak under water. Hence the imminence of the danger. The water
had increased from six inches to twenty. But if the leak could be
stopped, the boat could be baled out ; the bark once emptied, it
would rise once more to its normal water line, the fracture would be
out of water and thus dry, repair would be easy or, at least, possible.
Gilliatt, as we have already said, still had his carpenter's tools in
fairly good condition. But what uncertainty before reaching that
point ! what perils ! what evil chances ! Gilliatt heard the water
inexorably gushing in. A shock and all woidd sink. What
wretchedness ! Perhaps it was already too late. Gilliatt blamed
himself bitterly. He should have perceived the injury at once. The six
inches of water in the hold should have warned him. He had been
stupid to attribute those six inches to the rain and foam. He
reproached himself for having slept, for having eaten ; he almost
reproached himself with the tempest and the night. All was his fault.
These harsh self-reproaches were uttered as he went to and fro
about his work, and did not prevent his giving it careful attention.
The leak was found, that was the first step ; to stop it was the
second. Nothing more could be done for the moment. Carpentering
cannot be done under water. One favorable circumstance was that
the break in the hull had taken place in the space comprised
between the two chains
 178 THE TOILERS OF THE SEA. which stayed the smoke
stack on the starboard. The stuffing could be fastened to these
chains. Meanwhile, the water was gaining. The depth was now over
two feet. The water rofje above Gilliatt's knees.
 CHAPTER VI. DE PROFUNDIS AD ALTUM. GiLLiATT had at
his disposal, in the reserve of the rigging of the boat, a tolerably
large tarpaulin, provided with long lashings at its four corners. He
took this tarpaulin, fastened two corners of it by means of the
lashings to the two rings of the chains of the smoke stack, on the
side of the leak, and flung the tarpaulin overboard. The tarpaulin fell
like a table cloth between the little Douvre and the bark and sunk in
the waves. The pressure of water endeavoring to enter the hull
forced it against the hull and upon the hole. The more the water
pressed, the closer the tarpaulin clung. It was glued by the water
itself upon the fracture. The wound was dressed. This tarred canvas
interposed between the interior of the hold and the billows without.
Not another drop of water entered. The leak was covered, but not
calked. It was a respite. Gilliatt took the scoop and set to baling the
paunch. It was high time to lighten it. This work warmed him up a
little, but his fatigue was extreme. He was forced to admit to himself
that he should not be able to finish, and that he should not succeed
in baling out the hold. Gilliatt had hardly eaten, and he had the
humiliation of feeling himself exhausted. He gauged the progress of
his work by the fall of the level of the water to his knees. This fall
was slow. Moreover, the leak was only interrupted. The evil was
palliated, not repaired. The tarpaulin, thrust into the fract179
 130 '^^^ TOILERS OF THE SEA. ure by the water, was
beginning to form a tumor in the bull. It resembled a fist beneath
tliat canvas, seeking to burst it. The canvas, being solid and Avell
tarred, resisted; but the swelling and the tension were augmenting,
and it was not certain that the canvas would not give way, and the
tumor might burst at any moment. The irruption of the water would
recommence. In such a case, as ships' crews in distress are aware,
there is no other resource than stuffing. Rags are taken, of every
sort that comes to hand, everything that is known in the technical
language as '•' service," and as many as possible of them are thrust
into the crevice of the tarpaulin tumor. Of this " service," Gilliatt had
none. All the strips and oakum which he had stored up had either
been employed in his work or dispersed by the gale. At the most, he
might have found a few remains by ransacking the rocks. The boat
was sufficiently relieved to admit of his leaving it for a quarter of an
hour ; but how was such a search to be made without light ? The
darkness was complete. There was no longer any moon ; nothing
but the sombre, starry heavens. Gilliatt had no dry rope wherewith
to make a wick, no tallow to make a candle, no fire to light it, no
lantern to screen it. All was confused and indistinct in the bark and
on the reef. The water could be beard swashing against the
wounded hull ; but not even the hole was visible ; it was with his
hands that Gilliatt discovered the increasing tension of the tarpaulin.
Impossible to prosecute, in such darkness, a faithful search for the
bits of canvas and cordage scattered among the rocks. How was he
to glean these fragments when he covild not see clearly ? Gilliatt
gazed sadly at the night. All those stars, and no candle. The liquid
mass within the bark having diminished, the pressure from without
was increased. The swelling of the tarpaulin grew larger. It grew
more and more distended. It was like an abscess ready to burst. The
situation, improved for a moment, had again become threatening.
 DE PEOFUNDIS AD ALTUM. IgJ A plug was absolutely
necessary. Gilliatt had nothing but his clothes. He had placed them
to dry, as it will be remembered, on the salient rocks of the little
Douvre. He went and gathered them up and placed them on the rail
of the paunch. He took his tarpaulin coat, and kneeling in the water,
he thrust it into the crevice, pressing back the swelling of the
tarpaulin outwards, and, consequently, emptying it. To the tarpaulin
he added the sheepskin ; to the sheepskin, his woolen shirt ; to his
shirt, his pea-jacket. Everything went the same way. He had on but
one garment, he took this off, and with his trousers he enlarged and
strengthened the plugging. The stopper was made and seemed to
answer the purpose. This plug extended beyond the edge of the
breach with the tarpaulin for its envelope. The water, desirous of
entering, pressed against the obstacle, spread it out usefully on the
fracture, and consolidated it. It was a sort of exterior compress. In
the interior, the centre alone of the swelling having been thrust back,
there remained all around the breach of the crevice and of the plug
a circular pad of the tarpaulin, all the more adherent because the
very inequalities of the fracture retained it. The leak was stopped.
But nothing could be more precarious. Those sharp projections of
the fracture which fastened the tarpaulin might pierce it, and
through those holes the water would enter again. Gilliatt would not
oven perceive it in the darkness. It was hardly probable that that
plug would last until daylight. Gilliatt's anxiety assumed a different
form, but he felt it increasing at the same time that he felt his
strength decreasing. He set to baling the hold again, but his
exhausted arms could hardly lift the scoop full of water. He was
naked and shivering. Gilliatt felt the sinister approach of the end.
 182 THE TOILERS OF THE SEA. One possible chance
crossed his mind. Perhaps there was a sail in the offing. A fisherman
who should happen to be passing through the Douvres Avaters,
might come to his assistance. The moment had arrived when an
assistant was absolutely necessary. With a man and a lantern all
might be saved. Two of them could easily bale out the hold ; as soon
as the bark was water tight and no longer overflooded, she would
rise, she would regain her water line, the crevice would be above the
water, repairs could be made, the plug could immediately be
replaced by a section of planking, and the provisional apparatus
placed on the fracture by definite repairs, If not, he must wait until
daybreak, wait all night ! Fatal delay which might prove ruinous.
Gilliatt was in a fever of haste. If, by chance, some ship's lantern
was in sight, Gilliatt could make signals from the summit of the
Douvres. The weather was calm, there was no wind, a man moving
against the starry background of the sky stood a chance of being
noticed. The captain of a ship, or even the skipper of a fishing boat
does not sail the waters of the Douvres by night without pointing his
glass at the reef ; by way of precaution. Gilliatt hoped that he might
be seen. He scaled the wreck, grasped the knotted rope, and
ascended the grand Douvre. Not a sail on the horizon, not a light.
The water was deserted, as far as he could see. No assistance
possible, and no resistance possible. Gilliatt felt himself disarmed, a
thing which had not happened with him up to that moment. Dark
fatality was now his master. He, with his boat, with the engine of the
Durande, with all his labor, with all his success, with all his courage,
he belonged to the gulf. He had no longer any means of continuing
the struggle ; he became passive. How was he to prevent the tide
coming, the water rising, the night continuing ? That ])lug was his
only reliance. Crilliatt had worn himself out and stripped himself to
make
 DE PROFUNDIS AD ALTUM. 183 it, and complete it ; he
could neither fortify it nor render it firmer ; such as the plug was it
must remain, and fatally, all his effort having come to an end. The
sea had at its discretion that hasty apparatus applied to the leak.
How would that inert thing behave ? It was now it which was
combating, it was no longer Gilliatt. It was that rag, it was no longer
that mind. The swelling of the waves was sufficient to reopen the
fracture. More or less pressure ; the whole question lay there. All
was going to be solved by a blind struggle between two mechanical
quantities. Gilliatt could henceforth neither aid the auxiliary nor stop
the enemy. He was no longer anything but spectator of his life or his
death. That Gilliatt, who had been a providence, was, at the
supreme moment, replaced by an inanimate resistance. None of the
trials and terrors which Gilliatt had undergone approached this one.
On arriving at the Douvres reef, he had beheld himself surrounded
and seized, as it were, by solitude. This solitude did more than
environ, it enveloped him. A thousand menaces had shaken their
fists at him simultaneously. The wind was there, ready to blow ; the
sea was there, ready to roar ; impossible to stop that mouth, the
wind; impossible to deprive of its teeth that monster, the sea. Yet he
had struggled; a man, he had contended hand to hand with the
ocean ; he had wrestled with the tempest. He had held his own
against still other anxieties and necessities. He had become familiar
with all manner of distress ; without tools, he had been obliged to
perform great works ; without aid, to move burdens ; without
science, to solve problems ; without provisions, to eat and drink ;
without bed or roof, to sleep. On that reef, a tragic rack, he had
been put to the question in turn by the diverse torturing fatalities of
Nature : a mother when it seems good to her, an executioner when
she chooses.
 184 THE TOILERS OF THE SEA. He had conquered
isolation, conquered hunger, conquered thirst, conquered cold,
conqiiered fever, conquered work, conquered sleep. He had
encountered objects in coalition to bar his passage. After privation,
the elements ; after the sea, the tempest ; after the tempest, the
octopus ; after the monster, the spectre. Melancholy final irony. In
that reef, whence Gilliatt had reckoned on emerging in triumph,
Clubin, dead, came to gaze upon him with a mocking laugh. The
sneer of the spectre was justified. Gilliatt beheld himself lost. Gilliatt
beheld himself as dead as Clubin. Winter, famine, fatigue, the wreck
to dismember, the engine to tranship, the blows of the equinox, the
wind, the thunder, the octopus, — all these were nothing as
compared to the leak. Against the cold one can use, and Gilliatt had
used, fire ; against hunger, the shell fish from the rocks ; against
thirst, rain ; against the difficulties of salvage, industry and energy ;
against the sea and the storm, the breakwater ; against the octopus,
his knife ; — against a leak, nothing. The hurricane left him this
sinister farewell. A last reprisal, a traitorous thrust, the underhand
attack of the conquered on the conqueror. The tempest in its flight
shot this arrow to the rear. Defeats turned about, and dealt a blow.
It was the underhand stab of the abyss. One can combat with the
tempest, but how combat a leaking ? If the plug yielded, if the leak
opened again, nothing could prevent the boat from sinking. It was
the ligature on the artery unloosened. And the paunch once at the
bottom of the water with that heavy load, the engine, there would
be no means of raising it. This heroic effort of two Titanic months,
was ending in annihilation. To begin anew, was impossible. Gilliatt
had no longer either forge or materials. Perhaps at daybreak, he
would behold his whole work sink slowly and irremediably into the
gulf.
 BE PliOFUNDlS AD ALTUM. 185 It is a frightful thing to feel
sombre force beneath one. The gulf drew him to itself. His boat
submerged, nothing would be left to him but to die of cold and
hunger as that other had done, the shipwrecked sailor of " the Man "
rock. For two long months, the intelligences and providences which
are in the invisible had been witnesses of this : On the one hand, the
expanse, the waves, the winds, the lightnings, the meteors ; on the
other, a man. On the one side, the sea ; on the other, a soul. On the
one side, the infinite ; on the other, an atom. And there had been a
battle. And behold, perhaps this marvel had come to naught. Thus
ended in impotence this unprecedented deed of heroism ; thus
ended in despair this formidable combat, the struggle of nothing
against everything, this Iliad of one. Gilliatt gazed into space in
despair. He had no longer even a garment. He was naked in the
presence of immensity. Then, in the despondency of all that
unknown vastness, no longer knowing what was wanted of him;
confronting the gloom : in the presence of that impenetrable
obscurity ; in the uproar of the waters, the billows, the waves, the
surges, the foams, the squalls ; beneath the clouds, beneath the
gusts, beneath the vast scattered force ; beneath that mysterious
firmament of wings, of stars, of tombs ; beneath the possible
meaning mingled with vast things ; having around him and beneath
him the ocean, and above him the constellations ; — beneath the
unfathomable, he gave way, he renounced all, he flung himself flat
upon his back on the rock, Avith his face to the stars, vanquished,
and, clasping his hands before the terrible profundity, he cried to the
infinite : "Mercy ! " Hurled to earth by immensity, he prayed to it. He
was there alone, in that darkness, on that rock, in the midst of that
sea, overcome by exhaustion, resembling a man who has been
struck by lightning naked as a gladiator in the
 186 THE TOILERS OF THE SEA. circus ; only, instead of the
arena having the abyss ; instead of wild beasts, shadows ; instead of
the eyes of the populace, the gaze of the unknown ; instead of
vestals, stars ; instead of Caesar, God. It seemed to him, that he felt
himself dissolving in cold, fatigue, weakness, in prayer, in the gloom,
and his eyes closed '..■iW.
 CHAPTER Vri. THERE IS AN EAR IN THE UNKNOWN.
Several hours elapsed. The sun rose, dazzling. Its first ray lighted up
a motionless form upon the plateau of the great Douvre. It was
Gilliatt. He still lay outstretched on the rock. That icy and stiffened
nudity no longer even shivered. His closed eyelids were pallid. It
would have been difficult to say whether he was not a corpse. The
sun seemed to gaze upon him. If this naked man were not dead, he
was so near it that the slightest cold wind was sufficient to put an
end to him. The wind began to blow warm, and vivifying ; the
springlike breath of May. Meanwhile, the sun mounted higher in the
deep blue sky ; its less horizontal rays grew crimson. Its light
became heat. It enveloped Gilliatt. Gilliatt did not stir. If he breathed,
it was with that feeble, dying respiration which would hardly cloud a
mirror. The sun continued its ascent, its rays falling less and less
oblique upon Gilliatt. The wind, which had at first been merely
warm, was now hot. That rigid and naked body still remained devoid
of movement ; but the skin seemed less livid. The sun, as it
approached the zenith, fell perpendicularly on the plateau of the
Douvre. A prodigality of light was 187
 188 THE TOILERS OF THE SEA. poured from the heights of
heaven ; the vast reflection of the glassy sea was added to it ; the
rock began to grow warm, and revived the sleeper. A sigh heaved
Gilliatt's breast. He lived. The sun continued its caresses, which were
almost ardent. The wind, which was already the wind of the south
and of summer, approached Gilliatt like a mouth breathing gently.
Gilliatt stirred. The calmness of the sea was inexpressible. It
murmured like a nurse beside her child. The waves seemed to be
cradling the reef. The sea birds, who knew Gilliatt, flew above him
uneasily. This was no longer their former wild uneasiness. There was
something indescribably tender and fraternal about it. They uttered
little cries. They had the air of calling him. A sea-mew, who loved
him, no doubt, was so familiar as to come quite close to him. It
began to speak to him. He seemed not to hear. It jumped upon his
shoulder, and pecked gently at his lips. Gilliatt opened his eyes. The
birds, content and shy, flew away, Gilliatt sprang to his feet,
stretched like a lion awakened, ran to the edge of the platform, and
looked down into the passage between the Douvres beneath him.
The boat was there, intact. The plug had held firm ; the sea had
probably not disturbed it much. All was saved. Gilliatt was no longer
weary. His strength was renewed. His swoon had been a sleep. He
baled out the boat, made the hold dry, and raised the leak above the
water line, dressed himself once more, drank, ate, was joyous. The
leak, examined by daylight, required more labor than Gilliatt would
have believed. It was a rather serious injury. The whole day was not
too much for Gilliatt to repair it.
 THERE IS AN EAli IN THE UNKNOWN. 189 On the following
clay at dawn, after having removed the barriers and opened the exit
from the pass once more, clothed in his rags which had got the
better of the leak, wearing Clubin's belt with the seventy-five
thousand francs, standing erect upon the mended boat beside the
machinery which he had saved, Gilliatt left the Douvres reef with a
good wind and a propitious sea. He steered his course for Guernsey.
At the moment when he was quitting the reef, any one who had
been there might have heard him singing in a low tone the air of ''
Bonny Dundee."
 PART THIRD. — DERUCHETTE. BOOK FIRST. NIGHT AND
MOON. CHAPTER I. THE BELL OF THE PORT. The Saint-Sampson of
to-day is almost a city ; the Saint Sampson of forty years ago was
almost a village. When the spring had come and the long winter
evenings were over, people made short evenings there and went to
bed at nightfall. Saint-Sampson was an ancient curfew parish, having
preserved the habit of blowing out its candle early. People there
went to bed and rose with the day. These ancient Norman villages
imitate the habits of their fowls. Let us mention in addition, that
Saint-Sampson, with the exception of a few rich bourgeois families,
has a population of quarry-men and carpenters. The port is a port
for repairing. All day long they quarry stone or hew beams ; here the
pick, there the hammer. A perpetual handling of oak and of granite.
At night they are dropping with fatigue, and they sleep like lead.
Hea^^ work brings heavy slumbers. One evening in the beginning
of May, Mess Lethierry, after having, for several moments, gazed at
the moon through the trees, and listened to Deruchette's step, as
she walked alone in the cool of the evening, in the garden of les
Bravees, retired to his chamber, opening towards the port, and went
to bed. Douce and Grace were already in bed. Every one was asleep
191
 192 THE TOILEBS OF THE SEA. in tlie house except
Deruchette. Every one in Saint-Sampson was also asleep. Doors and
shutters were everywhere closed. There was no passing to and fro in
the streets. A few lights, like the blinking of eyes about to close,
gleamed redly here and there from the small windows in the roofs, a
sign of the retiring of the servants. It was some time since nine
o'clock had sounded from the old Eoman, ivy-draped clock-tower,
which shares with the church of Saint-Brelade in Jersey the
singularity of bearing as its date four ones, 1111, which signifies
"eleven hundred and eleven." Mess Lethierry's popularity in Saint-
Sampson was the result of his success. Success gone, a void had
resulted. We are forced to believe that bad luck is contagious, and
that people who are not fortunate have the plague, so speedily are
they placed in quarantine. The handsome sons of good families
avoided Deruchette. The isolation around les Bravees was now such
that they had not even learned in the house the little scrap of local
news which had set Saint-Sampson in commotion that day. The
rector of the parish, the Reverend Joe Ebenezer Caudray, was rich.
His uncle, the magnificent Dean of St. Asaph, had just died in
London. The news had been brought by the mail-sloop "Cashmere,"
which had arrived from England that very morning, and the mast of
which could be seen in the roadstead of Saint-Pierre-Port. The
"Cashmere " was to set out for Southampton the next day at noon
and, it was said, was to carry with it the reverend rector, recalled to
England in haste to be present at the official opening of the will, not
to mention the other pressing demands in connection with taking
possession of a great inheritance. All day long, Saint-Sampson had
discussed in a confused way, — the " Cashmere," the Reverend
Ebenezer, his dead uncle, his wealth, his departure, his possible
promotions in the future, had formed the burden of the buzzing. A
single house, not being informed, had remained silent, — les
Bravees. Mess Lethierry had thrown himself into his hammock all
dressed.
 THE BELL OF THE PORT. 193 Flinging himself into liis
hammock had been his resource ever since the wreck of the
Durande. Stretching out on hi? pallet is something to which every
prisoner has recourse, and Mess Lethierry was the prisoner of his
grief. He went to bed ; it was a truce, a space for taking breath, a
suspension of ideas. Did he sleep ? ISTo. Did he keep watch ? No.
Properly speaking, for the last two months and a half, — two months
and a half had elapsed since the disaster, — jMess Lethierry had
been like a somnambulist. He had not yet recovered possession of
himself. He was in that mixed and confused state which those are
acquainted with who have undergone great affliction. His reflections
were not thought, his sleep was not repose. During the day, he was
not fully awake. At night, he was not asleep. He was up and had
gone to bed; that was all. When he was in his hammock, oblivion
came to him a little ; he called this sleeping ; chimseras floated over
him and within him ; the nocturnal cloud, filled with confused faces,
traversed his brain ; the Emperoi Napoleon dictated his memoirs to
him ; there were several Deruchettes ; queer birds were in the trees
; the streets of Lons-le-Saulnier became serpents. Nightmare was
the respite from despair. He passed his nights in dreaming, and his
days in reverie. He sometimes remained a whole afternoon
motionless at his window, — which, as the reader will remember,
opened on the port, — with his head bent, his elbows on the stone,
his ears resting on his fists, his back turned to the whole world, his
eyes fixed on the old iron ring fastened in the wall of his house, to
which the Durande had formerly been moored. He was watching the
rust collect on that ring. Mess lethierry was reduced to the
mechanical function of living. The masi valiant of men, on being
deprived of their realizable idea, come to that pass. It is the result of
an existence vvhich has been rendered void. Life is the voyage, the
I'lea if; the itinerary. Where there is no longer an itinerary,
 194 ^^^ TOILERS OF THE SEA. one stops short. The goal
is lost, force is dead. Fate holds an obscure discretionary power. It
can touch even our moral being with its wand. Despair is almost the
destitution of the soul. Very great spirits alone resist. And still. Mess
Lethierry meditated continually, if absorption can be called
meditation, at the base of a sort of precipice of troubles.
Heartbroken words escaped him, like the following : " All that
remains for me is to ask from above my ticket of departure." Let us
note a contradiction in this nature, as complex as the sea, of which
Lethierry was, so to speak, the product: Mess Lethierry did not pray.
To be powerless is in itself a kind of force. In the presence of our
two great blindnesses, destiny and nature, — it ia in his
powerlessness that man has found his chief support, prayer. Man
makes his terror lend him succor ; he demands aid of his fears ;
anxiety counsels him to kneel. Prayer is an enormous force, peculiar
to the soul, and of the same species as mystery. Prayer addresses
itself to the magnanimity of the shadows ; prayer looks at the
mystery with the very eyes of the shadow, and before the powerful
intentness of that suppliant gaze, one feels that it is possible to
disarm the Unknown. This possibility once realized is a consolation in
itself. But Mess Lethierry did not pray. While he was happy, -God
existed for him, in flesh and blood, one might say ; Lethierry spoke
to him, pledged his word to Him, almost shook hands with Him from
time to time. But in Lethierry's unhappiness, God was eclipsed, a not
infrequent phenomenon. This happens when one has made for
himself a good God, who is a good fellow. In the state of mind in
which Lethierry then was, there existed for Lethierry but one clear
vision, Deruchette's smile. Beyond that smile, all was dark. For some
time past, no doubt on account of the loss of the Durande, the
counter shock of wliich she felt, this charming
 THE BELL OF THE POET. 195 smile of Deruchette's had
become more rare. She appeared thoughtful. Her birdlike and
childlike pretty ways had disappeared. She was no longer seen to
make a courtesy in the morning at the sound of the daybreak gun,
and say to the rising, sun ''Boom! Day, Pray take the trouble to
enter." She wore a very serious air at times, a sad thing in this sweet
being. Nevertheless, she made an eifort to smile at Mess Lethierry,
and to divert him, but her gayety grew more tarnished from day to
day, and became covered with dust, like the wing of a butterfly
which has a pin through its body. Let us add that, either out of grief
for her uncle's grief, for there are such things as reflected sorrows,
or for other reasons, she seemed now to incline greatly towards
religion. In the days of the former rector, M. Jaquemin Herode, she
had only gone to church, as the reader knows, four times a year.
She was very assiduous there now. She never missed a service,
either on Sunday or on Thursday. The pious souls of the parish
beheld this amendment with satisfaction. For it is great good fortune
when a young girl, who runs so many risks from men, turns towards
God. This at least causes the poor parents to feel more at ease on
the score of love affairs. In the evening, whenever the weather
permitted, she strolled for an hour or two in the garden of les
Bravees. She was almost as pensive there as Mess Lethierry, and
always alone. Deruchette was the last to go to bed. This did not
prevent Douce and Grace from always keeping an eye upon her,
through that instinct for watching which is common to domestics ;
spying relieves the tedium of serving. As for Mess Lethierry, in that
abstracted state of his mind, these little alterations in Deruchette's
habits escaped him. Moreover, he had not been born a duenna. He
did not even notice Deruchette's punctual attendance on the parish
services. Always tenacious in his prejudices against things and
people pertaining to the clergy, he would have viewed this
frequentation of church with no pleasure.
 196 THE TOILERS OF THE SEA. It was not because his own
moral condition was not in process of modification. Grief is a cloud
and changes its form. Eobust souls, as we have just said, are
sometimes, by certain blows of ill fortune, almost if not wholly,
thrown off their bearings. Virile characters, like Lethierry, react in a
given time. Despair has ascending degrees. From prostration one
mounts to despondency, from despondency to affliction, from
affliction to melancholy. Melancholy is a twilight. Suffering melts into
it in sombre joy. Melancholy is the happiness of being sad. These
mournful alternations were not made for Lethierry; neither the
nature of his temperament nor the character of his unhappiness
were adapted to these delicate shades. But at the moment when we
find him again, the reverie of his first despair had been, for about a
week, tending to dissipate ; without being less sad, Lethierry was
less inert ; he was still sombre, but he was no longer gloomy ; a
certain perception of facts and events had come back to him ; and
he had begun to experience something of that phenomenon which
may be called the re-entrance into reality. Thus, during the day, in
the lower room, he did not listen to the people's words, but he heard
them. Grace came in triumph one morning to tell Deruchette that
Mess Lethierry had broken the wrapper of a newspaper. This half
acceptance of reality is, in itself, a good symptom. It is
convalescence. Great misfortunes stun. It is in this way that one
emerges from the shock. But this amelioration first produces an
aggravation of the evil. The previous state of dreaminess blunted the
suffering ; one saw indistinctly ; one felt but little ; now the vision is
clear, one escapes nothing, one bleeds at everything. Pain is
accentuated by all the details which one perceives. One beholds all
again in memory. To remember all is to regret all. There are all sorts
of bitter after tastes in this return to the real. One is better and, at
the same time, worse. This was Lethierry's experience. His sufferings
were more distinct.
 THE BELL OF THE PORT. 197 It was a shock which had
restored Mess Lethierry to the sense of reality. Let us describe what
this shock was. One afternoon about the fifteenth or twentieth of
April, tho two knocks which announced the postman, had been
heard at the door of the big room of les Bravees. Douce had opened
the door. It was, in fact, a letter. This letter came from the sea. It
was addressed to Mess Lethierry. It was postmarked Lisboa. Douce
had carried the letter to Mess Lethierry, who was shut up in his
chamber. He had taken the letter, placed it mechanically on the
table, and had not glanced at it. This letter remained a full week on
the table, with its seal unbroken. But one morning it chanced that
Douce said to Mess Lethierry, — " Monsieur, shall I brush the dust
off from your letter ? " Lethierry appeared to wake up. " All right,"
said he. And he opened the letter. He read as follows, — " At Sea,
March 10. "Mess Lethierry, of Saint-Sampson: "You will be glad to
receive tidings of me. I am aboard the ' Tamaulipas,' bound for
Nevercomeback. Among the crew there is a sailor. Abler Tostevin, of
Guernsey, who will go back, and who will have some things to tell
you. I take advantage of our speaking the ship ' Hernan Cortes,'
bound for Lisbon, to send you this letter. " Be astonished, I am an
honest man. *' As honest as Sieur Clubin, "I am bound to believe
that you know what has occurred; nevertheless, it may not be
superfluous for me to inform you. " Here it is. " I have restored your
money to you. " I borrowed from you, somewhat irregvilarly, fifty
thousand francs. Before leaving Saint-Malo, I handed to your
confidential man, Sieur Clubin, three bank notes of a thousand
pounds each, making seventy-five thousand francs. You will, no
doubt, find this reimbursement sufficient.
Welcome to our website – the ideal destination for book lovers and
knowledge seekers. With a mission to inspire endlessly, we offer a
vast collection of books, ranging from classic literary works to
specialized publications, self-development books, and children's
literature. Each book is a new journey of discovery, expanding
knowledge and enriching the soul of the reade

Our website is not just a platform for buying books, but a bridge
connecting readers to the timeless values of culture and wisdom. With
an elegant, user-friendly interface and an intelligent search system,
we are committed to providing a quick and convenient shopping
experience. Additionally, our special promotions and home delivery
services ensure that you save time and fully enjoy the joy of reading.

Let us accompany you on the journey of exploring knowledge and

personal growth!

ebookname.com