Biochemistry III: Structural & Molecular Biology

Dr Blagojce (BJ) Jovcevski

Week 3 - Lecture 1
Protein Lifecycle I

Hospital Research Foundation Research Fellow

Discipline of Molecular & Biomedical Science
School of Biological Sciences

Room 1.49 MLS

[email protected]

BIOCHEMISTRY III - MOLECULAR & STRUCTURAL BIOLOGY 2024 1

Themes for the week
▪ Protein Structure (brief review)
▪ Protein Folding
▪ Folding in vitro

▪ Folding in vivo with chaperones

▪ Bacterial (Trigger Factor, Hsp70, GroEL/GroES)
▪ Eukaryotic (Ribosome-associated complex, Hsp70, TRiC)

▪ Protein Misfolding
▪ Protein Lifecycle

BIOCHEMISTRY III - MOLECULAR & STRUCTURAL BIOLOGY 2024 2

Protein structure

BIOCHEMISTRY III - MOLECULAR & STRUCTURAL BIOLOGY 2024 3

Protein structure

BIOCHEMISTRY III - MOLECULAR & STRUCTURAL BIOLOGY 2024 4

 Protein folding patterns - motifs
• A motif is a recognisable folding pattern involving two or more elements of secondary
structure and the connections between them
• Larger motifs can be constructed of smaller ones
• Protein tertiary structure is more reliably conserved than is amino acid sequence

β-α-β loop

globin fold

Β-barrel

BIOCHEMISTRY III - MOLECULAR & STRUCTURAL BIOLOGY 2024 5

 Protein folding patterns - domains
• Domains are part of a polypeptide chain that is independently stable or could undergo movements as a single
entity with respect to the entire protein
• Domains can appear as distinct globular lobes or extensive contacts can occur between domains and make individual
domains hard to discern
• Two or more domains of a protein can have different functions, which are coordinated overall function of the protein
small proteins usually only have one domain

calmodulin

BIOCHEMISTRY III - MOLECULAR & STRUCTURAL BIOLOGY 2024 6

Peptide bonds

Peptide bond is planar (double-bond character)

Phi (Φ) and Psi (ψ) angles can vary; their rotation allows polypeptides to
adopt their various structures (alpha-helices, beta-sheets, etc.). In a fully
extended structure these are 180 degrees.

BIOCHEMISTRY III - MOLECULAR & STRUCTURAL BIOLOGY 2024 7

Peptide bonds

potential for steric hindrance

Cis conformation is rare (except for proline)

BIOCHEMISTRY III - MOLECULAR & STRUCTURAL BIOLOGY 2024 8

Bonding interactions

+ protein-solvent
interactions

BIOCHEMISTRY III - MOLECULAR & STRUCTURAL BIOLOGY 2024 9

Protein synthesis on ribosome

BIOCHEMISTRY III - MOLECULAR & STRUCTURAL BIOLOGY 2024 10

Structural view of protein lifecycle

BIOCHEMISTRY III - MOLECULAR & STRUCTURAL BIOLOGY 2024 11

 First protein folding experiments – Anfinsen
• Christian B. Anfinsen – RNAse A (Nobel Prize, 1972)
• Folding is encoded in the amino acid sequence
• Native state is the minimum energy state

dilution
β-mercaptoethanol to
urea buffer

Native Unfolded Native

catalytically active state reduced disulfide bonds catalytically active state
catalytically inactive refolded correctly
1/105 random chance
Anfinsen, CB (1973) Principles that govern the folding of protein chains. Science 181, 223-230.
http://nobelprize.org/nobel_prizes/chemistry/laureates/1972/anfinsen-lecture.html

BIOCHEMISTRY III - MOLECULAR & STRUCTURAL BIOLOGY 2024 12

Protein folding problem
• Leventhal’s Paradox:
• Assume a chain of 100 amino acids, allow only 3 conformations
• Possible conformations = 3100 ~ 1048

• Assume bond rotation rate 1014 sec

• Reaching the native state would take 1026 years
(longer than the age of the universe!)

Can not be a random walk

BIOCHEMISTRY III - MOLECULAR & STRUCTURAL BIOLOGY 2024 13

Protein folding thermodynamics
• Each point on curve represents a different polypeptide conformation
• Each conformation has a different ΔG
• Curve is only one of many possible folding paths

unfolded

DG intermediate

native

number of internal contacts

compactness
coverage of hydrophobicity

BIOCHEMISTRY III - MOLECULAR & STRUCTURAL BIOLOGY 2024 14

Protein folding funnel
• A new view of protein folding suggests there is no single route, but a large ensemble of structures follow a
many dimensional funnel to native structure

Limited number of secondary

structure elements: helices,
sheets and turns

Folding can be thought

to occur along
“energy surfaces or landscapes”

BIOCHEMISTRY III - MOLECULAR & STRUCTURAL BIOLOGY 2024 15

Energy of protein folding
• Two major contributions to energy difference between unfolded and folded state (enthalpy and
entropy)
• Enthalpy (H) is increased upon folding (hundreds kcal/mol)
• Noncovalent interactions are maximised
• Entropy is decreased upon folding (hundreds kcal/mol)
• Causes one main conformation = highly ordered structure

• Difference in energy (free energy, ΔG) between folded (native) and unfolded (denatured) state is
small = 1-15 kcal/mol
• Small ΔG is necessary
• Too large a free energy change would mean a very stable protein, However, structural flexibility is important
to protein function and degradation

BIOCHEMISTRY III - MOLECULAR & STRUCTURAL BIOLOGY 2024 16

Overview of energy

Strength
Bond
(kcal/mole)
H-bonds 3-7
Ionic bonds 10
Hydrophobic
1-2
interactions
Van der vaals
1
interactions
Disulfide bridge 51

BIOCHEMISTRY III - MOLECULAR & STRUCTURAL BIOLOGY 2024 17

The hydrophobic effect
• Important for folding - every amino acid participates!
Experimentally determined hydrophobicity levels
2.25 Trp 0.26 Thr

1.80 Ile 0.13 His

1.79 Phe 0.00 Gly

1.70 Leu -0.04 Ser

1.54 Cys -0.22 Gln

1.23 Met -0.60 Asn

1.22 Val -0.64 Glu

0.96 Tyr -0.77 Asp

0.72 Pro -0.99 Lys

0.31 Ala -1.01 Arg

BIOCHEMISTRY III - MOLECULAR & STRUCTURAL BIOLOGY 2024 18

Mechanisms of protein folding
• Folding pathway can vary between proteins
• Single energy barrier with one pathway or
• Single folding pathway that has sequential transitional states that have limited flexibilities along the pathway or
• Have multiple transition states with similar energy values and a variety of pathways to get to the final folded
state

• Removal of hydrophobic sidechains from contact with water is driving force for folding (occurs early)

• It is unlikely that there is a single mechanism for protein folding

BIOCHEMISTRY III - MOLECULAR & STRUCTURAL BIOLOGY 2024 19

Classic models: Framework
• Elements of native local secondary structure could form independently of tertiary structure
• These elements would diffuse until they collided, successfully adhering and coalescing to give the tertiary
structure (diffusion-collision model)

BIOCHEMISTRY III - MOLECULAR & STRUCTURAL BIOLOGY 2024 20

Classic models: Nucleation
• Some neighbouring residues form native secondary structure that act as a nucleus from which the
native structure would propagate, in a stepwise manner
• Tertiary structure forms as a necessary consequence of the secondary structure

BIOCHEMISTRY III - MOLECULAR & STRUCTURAL BIOLOGY 2024 21

Classic models: Hydrophobic collapse
• The protein collapses rapidly around its hydrophobic sidechains and then rearranges from restricted
conformational space occupied by the intermediate
• Secondary structure would be directed by native-like tertiary structure

BIOCHEMISTRY III - MOLECULAR & STRUCTURAL BIOLOGY 2024 22

Molten globule state
• Protein folding can occur via a series of partially folded intermediates
• Compact globule, yet expanded over a native radius (~10-30%)
• Native-like secondary structure, can be measured experimentally
• Limited tertiary structure, highly dynamic
• Exposed hydrophobic core
• May be present only transiently

BIOCHEMISTRY III - MOLECULAR & STRUCTURAL BIOLOGY 2024 23

Protein denaturants
• Temperature
• High temperature causes protein unfolding, aggregation
• Some proteins are sensitive to cold denaturation
• pH
• Net charge leads to intramolecular charge-charge repulsion

• Heavy metals (e.g., lead, cadmium, etc.)

• Highly toxic; efficiently induce the ‘stress response’

• Proteotoxic agents (e.g. alcohols, cross-linking agents, etc.), oxygen radicals,

ionising radiation
• Cause permanent protein damage

• Chaotropes (urea, guanidine hydrochloride, etc.)

• Highly potent at denaturing proteins , often used in protein folding studies
• Preferential solvation of the unfolded state, involving predominantly hydrophobic
related properties, and to a lesser extent H-bonding

BIOCHEMISTRY III - MOLECULAR & STRUCTURAL BIOLOGY 2024 24

Protein stability
• The net stability of a protein is defined as the difference in free energy between the native and
denatured state:
• Both GN and GU contribute to G
• Decreasing the energy of the folded state or increasing the energy of the unfolded state have the same effect
on ΔG

• The free energy may be readily calculated from the following relationships:

K = [N]/[U] = FN/(1- FN)

FN = fraction folded

BIOCHEMISTRY III - MOLECULAR & STRUCTURAL BIOLOGY 2024 25

Following acquisition of structure
• Gain of 2°, 3° and 4° structures
• Circular dichroism
• Fluorescence measurements
• Size determination (e.g., native gel electrophoresis, size exclusion chromatography, protease sensitivity assays, etc)
• NMR

• Gain of activity
• enzymatic or other

denaturation renaturation native

structure?

BIOCHEMISTRY III - MOLECULAR & STRUCTURAL BIOLOGY 2024 26

Measuring protein stability
• Any methods that can distinguish between U and F
• Absorbance (e.g. Trp, Tyr)
• Fluorescence (Trp)-difference in emission max and intensity
• CD (far or near UV)
• NMR
• Calorimetry
• Urea gradient gels - difference in the migrating rates
between F and U
• Catalytic activity
• Chromophoric/fluorophoric probes

BIOCHEMISTRY III - MOLECULAR & STRUCTURAL BIOLOGY 2024 27

Challenges in studying protein folding
• Folding can be very fast, millisecond to second
• Small energy changes between the denatured state to the native state (1-15 kcal/mol)
• Equivalent to the strength of a few hydrogen bonds

• The states populated along pathway are ensembles of structures

BIOCHEMISTRY III - MOLECULAR & STRUCTURAL BIOLOGY 2024 28

Protein folding – key concepts
• In the equilibrium between the denatured state (unfolded or partially unfolded) and the native state
(folded, biologically functional), under physiological conditions most molecules are in the native state
• Primary structure determines higher structure
• Many proteins can fold without assistance from cellular components
• Some require assistance from chaperones (but not folding ‘instructions’)

• Proteins can be unfolded and refolded

• Proteins fold on a defined pathway (or a small number of alternative pathways)
• They don't randomly search all possible conformations until they arrive at the most stable (lowest free energy)
structure

BIOCHEMISTRY III - MOLECULAR & STRUCTURAL BIOLOGY 2024 29

Protein folding view

BIOCHEMISTRY III - MOLECULAR & STRUCTURAL BIOLOGY 2024 30

Next time…
▪ Protein folding in vivo

BIOCHEMISTRY III - MOLECULAR & STRUCTURAL BIOLOGY 2024 31