Chapter 10: Membrane Structure

Cell membrane overview

Function: Cell membranes are vital for cellular life. They de ne cell boundaries and maintain di erences between
cytoplasm and extracellular space.
Eukaryotic Cells: These contain internal membranes (nuclear membrane, ER, Golgi apparatus, mitochondria) which
create distinct environments within the cell.
Ion Gradients: Specialized membrane proteins establish ion gradients, which are critical for ATP synthesis, solute
transport, and generating electrical signals in nerve/muscle cells.
Signal Reception: Proteins in the plasma membrane act as sensors for external signals, altering cell behavior
according to environmental changes.
Structure: All biological membranes share a similar structure: a thin lm of lipids and proteins held together by non-covalent interactions.

Membrane Structure
Fluid Mosaic Model
• Dynamic Nature: Membranes are uid structures where molecules move within the plane.
• Lipid Bilayer: Composed of lipids arranged in double layers (about 5 nm thick), serving as barriers to most water-soluble substances.
• Protein Functions:
◦ Transport of speci c molecules
◦ Catalyzing membrane-associated reactions (like ATP synthesis)
◦ Transmembrane protein : Structural connections between cytoskeleton, matrix, or other cells.
◦ Serve as receptors to detect and transduce chemical signals
◦ Estimated that 30% of proteins coded in an animal genome are membrane proteins.

Lipids in Cell Membranes

→ provides the basic structure for all cell membranes
→ easily seen by electron microscopy

• Major Lipid Types:

◦ Phospholipids: Main component; hydrophilic
head + two hydrophobic tails.
◦ Sphingolipids: Built from sphingosine;
important for membrane structure.
◦ Cholesterol: Strengthens the membrane and
a ects uidity.
• Lipid Composition: Lipid molecules constitute about 50% of
the mass of most animal cell membranes, nearly all of the
remainder being protein.
◦ Approximately 5 × 10^6 lipid
molecules in a 1 μm × 1 μm area of
lipid bilayer.
◦ About 10^9 lipid molecules in the
plasma membrane of a small animal
cell.
◦ All lipid molecules are amphiphilic,
having a hydrophilic or polar end and
a hydrophobic or nonpolar end.

• Phospholipid Structure:
◦ Phosphoglycerides: Glycerol backbone, two fatty acid tails, phosphate group.
◦ Fatty Acids: Vary in length (14 to 24 carbons), can be saturated or unsaturated, a ecting packing.

✴ Phospholipids: Most abundant membrane lipids with a polar head group, phosphate group, and two hydrophobic hydrocarbon tails.
◦ Tails are typically fatty acids, di ering in lengths
(14 to 24 carbon atoms).
◦ One tail usually has one or more cis-double
bonds (unsaturated), while the other tail is
saturated.
◦ Each cis-double bond creates a kink in the tail,
in uencing the packing and uidity of the
membrane.

✦ Phosphoglycerides:
- Main phospholipids in most animal cell
membranes;
- consist of a three-carbon glycerol backbone,
with 2 long-chain fatty acids linked through
ester bonds and the third carbon attached to
a phosphate group linked to various head
groups.

✴ Sphingolipids: a class of phospholipids built from

sphingosine (not glycerol).
Sphingosine: Long acyl chain with 1 amino grp (NH₂) and 2 hydroxyl grps (OH).
Sphingomyelin: The most common sphingolipid, consisting of:
• A fatty acid tail attached to the amino group.
• A phosphocholine group attached to the terminal hydroxyl group.
ff
fl
fl
fi
fl
ff
fl
fi
fi
ff
ff
 Phospholipids in mammalian cell membranes:
• Phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin together make up more than half the lipid mass in most
mammalian cell membranes

✴ Glycolipids: Similar to sphingolipids, but with sugars attached instead of a phosphate-linked head group.

✴ Cholesterol:
- Found in eukaryotic plasma membranes in large amounts (up to 1 molecule per phospholipid).
- A sterol with a rigid ring structure, a polar hydroxyl group, and a short non-polar hydrocarbon chain.
- Cholesterol molecules orient with their hydroxyl group near the polar head groups of adjacent
phospholipid molecules.

Hydrophilic molecules dissolve in water due to favorable electrostatic interactions or hydrogen bonds.
Hydrophobic molecules are insoluble in water, forming ordered water cages around them, increasing free
energy.
Hydrophobic parts of amphiphilic molecules cluster together, minimizing water interactions.
Bilayer formation: Amphiphilic molecules aggregate with hydrophobic tails inward and hydrophilic heads outward,
forming either spherical micelles or double-layered bilayers.
The self-sealing property of bilayers: Tears in the bilayer cause energetically unfavorable free edges, prompting lipids to
rearrange and seal the membrane.
Closed compartments are formed to avoid free edges, essential for bilayer stability.

Lipid Bilayer Fluidity: Lipid molecules can di use freely within the plane of the bilayer. This
was rst demonstrated in synthetic lipid bilayers, like liposomes (spherical vesicles) or
planar bilayers (across holes in partitions).
Techniques to measure motion:
•Lipids can be labeled with a uorescent dye or a gold particle on the polar head to track
individual molecule di usion.
•Alternatively, a spin label (e.g., nitroxide) can be added to the lipid head group to study
movement.

- Electron Spin Resonance (ESR) Spectroscopy: Detects motion and orientation of spin-labeled lipids in a bilayer by measuring their paramagnetic signal
(similar to NMR).
- Flip- op: Rare for phospholipids; occurs on a time scale of hours. Cholesterol can ip- op more rapidly.
- Lateral di usion: Rapid (~107 times per second), allowing lipids to exchange places within the monolayer.
Di usion coe cient (D) ~ 10^-8 cm²/sec; lipids di use across a bacterial cell (~2μm) in about 1 second.
Membrane Mobility: Lipid molecules in synthetic bilayers and biological membranes are in a two-dimensional liquid state, moving laterally but remaining
con ned to their own monolayer.
Phospholipid Synthesis Problem: Phospholipids are made in one monolayer (usually cytosolic). To ensure proper bilayer growth, ippases catalyze the ip-
op between monolayers.
Fusion Prevention: Liposomes do not spontaneously fuse due to hydration shells around the lipid head groups. This prevents uncontrolled fusion, maintaining
the compartmental integrity of membrane-enclosed organelles.

Lipid Bilayer Fluidity: Fluidity is crucial for membrane functions like transport and enzyme activity. It is regulated by both membrane composition and
temperature.
- Phase Transition:
Synthetic lipid bilayers transition from a liquid state to a rigid crystalline (gel) state at a characteristic temperature. This temperature is lowered by:
- Short hydrocarbon chains: Reduce interactions between tails, keeping the membrane uid.
- Cis-double bonds: Create kinks in the chains, preventing tight packing and maintaining uidity at lower temperatures.
Temperature Regulation in Organisms: Organisms like bacteria and yeasts adjust fatty acid composition to maintain uidity as temperature changes. At
lower temperatures, they increase cis-double bonds in fatty acids.

Cholesterol's Role:
• Enhances permeability-barrier properties by interacting with the hydrocarbon chains of
phospholipids.
• Tightens packing of lipids but does not reduce membrane uidity.
• Prevents crystallization of hydrocarbon chains at high concentrations, especially in eukaryotic
plasma membranes.
Membrane Composition:
• Bacterial plasma membranes: Composed mainly of one type of phospholipid, no cholesterol.
• Archaeal membranes: Have di erent lipid compositions, usually containing unique structures.

Phenyl Chains in Archaea: Archaea, especially thermophilic ones, use 20-25 carbon-long phenyl chains instead of fatty acids. These chains are hydrophobic
and exible, providing membrane stability, especially in high heat environments where the chains span both bilayer lea ets.

Eukaryotic Membranes: Eukaryotic plasma membranes are more complex than those of prokaryotes and archaea:
• large amounts of cholesterol.
• mixture of di erent phospholipids, increasing membrane diversity.
• Complex Lipid Composition:
• Eukaryotic membranes contain a wide variety of lipid species, ranging from 500 to 2000 species, with even simple membranes (e.g., red
blood cell plasma membranes) containing over 150.
• Complexity arises from variations in head groups, hydrocarbon chain lengths, and desaturation of phospholipids, as well as distinct
minor lipids.
fl
fi
fl
fi
fl
ff
ff
ff
ff
ffi
fl
ff
ff
ff
fl
fl
fl
fl
fl
fl
fl
fl
fl
• Inositol Phospholipids:
• Present in small amounts in animal cell membranes, they play key roles in membrane tra c and cell signaling.
• Their synthesis and degradation are regulated by enzymes, creating intracellular signaling molecules and lipid docking sites to recruit
speci c proteins.

Lipid bilayers are two-dimensional uids, so lipids are expected to mix randomly.
- Van der Waals forces between lipid tails are not strong enough to keep
phospholipids together in xed groups.
- In arti cial bilayers, speci c lipids can segregate into separate domains
(phase separation). There is debate about whether similar lipid segregation
occurs in living cells.
- Large-scale lipid phase separations are rarely observed in living
membranes.
- Instead, membrane proteins and lipids form temporary, dynamic clusters
through protein-protein interactions.These clusters can be nanometer-
sized or larger, such as caveolae involved in endocytosis.
- Lipid phase partitioning in arti cial bilayers may contribute to raft formation in cell membranes, organizing membrane proteins for transport.

Most cells store excess lipids in lipid droplets, which serve as a source for membrane synthesis or energy.
- Adipocytes (fat cells) specialize in lipid storage and contain a giant lipid droplet that occupies most of the cytoplasm.
- Other cell types have multiple smaller lipid droplets, whose number and size depend on metabolic state. Fatty acids can be released from lipid droplets
on demand and transported via the bloodstream. Lipid droplets store neutral lipids (triacylglycerols and cholesterol esters), synthesized in the
endoplasmic reticulum (ER).
- Since these lipids are hydrophobic and lack hydrophilic head groups, they form 3D droplets instead of bilayers. Unlike other organelles, lipid droplets are
surrounded by a single phospholipid monolayer, containing various proteins.
- Some of these proteins are enzymes involved in lipid metabolism, but the function of most remains unknown.
Lipid droplets form quickly in response to high fatty acid levels, likely originating from speci c ER regions rich in lipid metabolism enzymes.

The two monolayers of the lipid bilayer have distinct lipid compositions.

In human red blood cells, phosphatidylcholine and sphingomyelin are in the outer
monolayer, while phosphatidylethanolamine and phosphatidylserine are in the inner
monolayer.
Phosphatidylserine's negative charge creates a charge di erence between the two
bilayer halves.
Phospholipid translocators maintain this lipid asymmetry.

Lipid asymmetry plays a role in cell signaling, helping convert extracellular signals into intracellular ones.
- Protein kinase C (PKC) binds to phosphatidylserine in the cytosolic monolayer, requiring its negative charge for activation.
- Phosphatidylinositol (PI) can be phosphorylated by lipid kinases (e.g., PI 3-kinase), creating binding sites that recruit intracellular signaling proteins.
- Phospholipases cleave phospholipids to produce intracellular signaling molecules; for example, phospholipase C generates fragments that activate PKC
and release Ca²⁺ from the ER.
- Apoptotic cells expose phosphatidylserine on their outer monolayer, signaling macrophages to digest them. This translocation occurs due to:
1. Inactivation of the phospholipid translocator, which normally keeps phosphatidylserine in the inner monolayer.
2. Activation of scramblase, which randomly redistributes phospholipids between monolayers.

Glycolipids are sugar-containing lipid molecules found exclusively in the monolayer facing away from the cytosol. In animal cells, glycolipids are derived from
sphingosine, like sphingomyelin. They tend to self-associate via hydrogen bonds (sugar groups) and van der Waals forces (hydrocarbon chains), often
clustering in lipid rafts. Their asymmetric distribution results from sugar addition in the Golgi lumen, making them topologically equivalent to the cell exterior.

Glycolipids constitute ~5% of plasma membrane lipids and are also present in
some intracellular membranes.

Gangliosides are the most complex glycolipids, containing oligosaccharides with

one or more sialic acid moieties, giving them a net negative charge.
Gangliosides are particularly abundant in nerve cells (5–10% of total lipids), but
present in smaller amounts in other cells.

Functions of glycolipids:
• Protection: Concentrated at apical surfaces of epithelial cells, shielding
them from harsh environments.
• Electrical e ects: Charged glycolipids, like gangliosides, in uence
membrane potential and ion concentrations (Ca²⁺).
• Cell recognition: Serve as binding sites for lectins, playing roles in cell-
cell adhesion.
• Nervous system health: Mice lacking complex gangliosides show axonal degeneration and reduced myelination.
fi
fi
ff
fi
fi
fi
fl
ff
fl
ffi
fi
Some bacterial toxins and viruses use glycolipids as entry points:
• Cholera toxin binds to GM1, leading to increased cAMP,
massive Cl⁻ e ux, and severe diarrhea.
• Polyomaviruses also use gangliosides for cell entry.

Membrane Proteins & Function:

- While the lipid bilayer provides structure, membrane proteins
perform most functional roles.
- These proteins de ne each membrane’s functional properties.

Protein Composition in Di erent Membranes:

- Myelin membrane (insulates nerve axons): <25% protein
- Mitochondria & chloroplast membranes (ATP production): ~75% protein.
- Plasma membrane: ~50% protein.

Lipid vs. Protein Ratio:

- Lipid molecules are smaller than proteins.
- Even in membranes that are 50% protein by mass, there are ~50 lipid molecules
per protein molecule.

Protein Structure & Association with Bilayer:

- Membrane proteins vary widely in structure and interaction with lipids, re ecting
diverse functions.

General Properties of Membrane Proteins:

• Amphiphilic: Have hydrophobic and hydrophilic regions.
• Associate with the lipid bilayer in various ways.
• Cell-surface receptors bind extracellular signals and generate intracellular responses.
• Membrane transport proteins (e.g., multipass transmembrane proteins,) create pathways for small hydrophilic molecules.
Transmembrane Proteins: Span the lipid bilayer, with parts exposed on both sides (Examples 1, 2, 3).
Hydrophobic regions interact with lipid tails inside the bilayer.
Hydrophilic regions are exposed to water on either side.
Some have a covalently attached fatty acid chain that increases hydrophobicity (Example 1).

Cytosolic Membrane Entirely in cytosol, attached to the bilayer by:

Proteins: • Amphiphilic α helix on protein surface (Example 4).
• Covalently attached lipid chains (Example 5).

Extracellular Membrane • Attached to the outer monolayer via an oligosaccharide-linked lipid anchor (Example 6).
Proteins: • These proteins are synthesized in the ER, where:
◦ The transmembrane segment is cleaved o .
◦ A glycosylphosphatidylinositol (GPI) anchor is added.
◦ Transport vesicles deliver them to the plasma membrane.

Peripheral Membrane Do not penetrate the hydrophobic bilayer (Examples 7, 8).

Proteins: Bind to the membrane via noncovalent interactions with other proteins.
Can be removed by gentle extraction (e.g., high/low ionic strength or extreme pH).

Single-Sided Membrane Proteins

• Associated with one monolayer or a protein domain on that side.
• Intracellular signaling proteins often anchored to the cytosolic membrane by covalently attached lipid groups(fatty
acid or prenyl groups.

Lipid Anchors & Signaling Proteins

• Myristic acid (C14 fatty acid) attaches to the N-terminal of Src family tyrosine kinases during synthesis.
• Palmitic acid (C16 fatty acid) attaches to a cysteine side chain, strengthening the membrane attachment.
• Palmitoylation is signal-dependent; removal allows proteins to return to the cytosol.
• Ras family GTPases use a prenyl + palmitic acid combo for membrane recruitment.

Transient Membrane Attachment

• Peripheral membrane proteins associate via regulated protein–protein interactions.
• Some proteins switch between soluble and membrane-bound states by:
◦ Conformational changes exposing a hydrophobic peptide or lipid anchor.
◦ Rab GTPases regulate intracellular membrane tra c:
▪ GDP-bound state → Soluble in cytosol.
▪ GTP-bound state → Expose lipid anchor, tethering to membranes.
• Dynamic membrane interactions expand functional versatility.
- Unique orientation due to asymmetric insertion into ER membrane.
- Cytosolic & noncytosolic domains have different functions.
Orientation & Structure - Membrane-spanning segments interact with the hydrophobic lipid bilayer.
- Peptide bonds are polar → form hydrogen bonds in absence of water.
- α-Helix formation maximizes hydrogen bonding (Figure 10–19).
- Single-pass proteins: Cross bilayer once.
Types of Transmembrane Proteins - Multipass proteins: Cross bilayer multiple times.
- β-Barrel structure: β-sheet rolled into a cylinder, seen in porin proteins.
ffl
fi
ff
ff
ffi
fl
 - X-ray crystallography provides 3D structure con rmation.
- Hydropathy plots predict α-helical transmembrane segments (20–30 hydrophobic amino acids).
Determining Membrane-Spanning Regions
- ~30% of an organism’s proteins are transmembrane proteins.
- β-Barrel segments (≤10 amino acids) cannot be detected by hydropathy plots.
- Polypeptides tend to pass fully through the bilayer before changing direction.
- Multipass proteins can have membrane-folded regions that do not interact with the lipid core.
Membrane Protein Folding & Functional Regions
- These folded regions form ion/water channel pores and determine substrate speci city.
- Cannot be identi ed by hydropathy plots, only by X-ray or electron crystallography.

Using hydropathy plots to localize potential α-helical membrane-spanning segments in a polypeptide chain. The free energy needed to transfer successive
segments of a polypeptide chain from a nonpolar solvent to water is calculated from the amino acid composition of each segment using data obtained from
model compounds. This calculation is made for segments of a xed size (usually around 10–20 amino acids), beginning with each successive amino acid in the
chain.
The “hydropathy index” of the segment is plotted on the Y axis as a function of its location
in the chain. A positive value indicates that free energy is required for
transfer to water (i.e., the segment is hydrophobic), and the value assigned is
an index of the amount of energy needed.
Peaks in the hydropathy index appear at the positions of hydrophobic segments
in the amino acid sequence. Glycophorin (A) has a single membrane-spanning α helix,
and one corresponding peak in the hydropathy plot. Bacteriorhodopsin (B) has seven
membrane-spanning α helices and seven corresponding peaks in the hydropathy plot.

Role of - Do not always contribute to protein folding on either side of the membrane.
Transmembrane - Can be engineered as soluble cytosolic or extracellular domains for structural studies.
α Helices - Play a role in stabilizing membrane protein structure and function.

Single-Pass - Often form homo- or heterodimers, held by noncovalent but strong interactions.
Transmembrane - Hydrophobic amino acid sequence directs protein–protein interaction in the membrane.
Proteins - Found in receptor proteins (e.g., growth factor receptors, GPCRs).

Multipass - Speci c folding positions determined by interactions between helices.

Transmembrane - Essential for ion channels, transporters, and enzyme complexes.
Proteins - Some helices shield other transmembrane helices from direct lipid contact.

Protein Folding - Initially inserted sequentially into the membrane by protein translocators.
& - Hydrophobic nature required as helices are rst surrounded by lipids.
Hydrophobicity - During nal folding, protein–protein interactions replace protein–lipid interactions.
- Ensures proper positioning and functional stability in the membrane.

β-Barrel Proteins and Large Channels

- Multipass membrane proteins with transmembrane segments arranged as β barrelsinstead of α helices.
General Structure of β-Barrel Proteins - Rigid structure, allowing them to crystallize easily for x-ray crystallography studies.
- The number of β strands varies from 8 to 22 per barrel.
- Common in the outer membranes of bacteria, mitochondria, and chloroplasts.
- Some β-barrel proteins function as pore-forming channels, allowing the passage of small hydrophilic
Distribution & Function
molecules.
- Porins are a well-studied class of β-barrel proteins.
- Many porins are formed from a 16-strand antiparallel β sheet rolled into a cylindrical structure.
- Polar amino acids line the inside of the channel, while nonpolar amino acids face the hydrophobic lipid
Porins & Selectivity bilayer.
- Loops protruding into the channel determine selectivity for speci c solutes.
- Maltoporin in E. coli preferentially allows maltose and maltose oligomers to cross.
- FepA transports iron ions across the bacterial outer membrane.
FepA: A Complex β-Barrel Transporter - Contains 22 β strands and a globular domain that lls the barrel’s interior.
- Iron ions bind inside the barrel, and their transport mechanism remains unclear.
- Some β-barrel proteins function as receptors or enzymes, rather than as transporters.
- These barrels are completely lled with amino acid side chains, preventing molecule passage.
Non-Transport β-Barrel Proteins
- The barrel serves as an anchor, holding the protein in the membrane and positioning cytosolic loops for
intracellular binding.
- Most eukaryotic and bacterial multipass membrane proteins use α-helices instead of β barrels.
- α-Helices can slide against each other, allowing conformational changes for ion channel gating, solute
Comparison with α-Helical Membrane
transport, and signal transduction.
Proteins
- In contrast, β-barrel proteins have rigid hydrogen bonds between β strands, making structural changes
highly unlikely.
fi
fi
fi
fi
fi
fi
fi
fi
fi
fi
β barrels formed from different numbers of β strands.(A) The E. coli
OmpA protein serves as a receptor for a bacterial virus. (B) The E. coli
OMplA protein is an enzyme (a lipase) that hydrolyzes lipid molecules.
The amino acids that catalyze the enzymatic reaction (shown in red)
protrude from the outside surface of the barrel. (C) A porin from the
bacterium Rhodobacter capsulatus forms a water- lled pore across the
outer membrane. The diameter of the channel is restricted by loops
(shown in blue) that protrude into the channel. (D) The E. coli FepA
protein transports iron ions. The inside of the barrel is completely lled
by a globular protein domain (shown in blue) that contains an iron-
binding site.

Many membranes are glycosylated:

Glycosylation - Most transmembrane proteins in animal cells are glycosylated.
of Membrane - Sugar residues are added in the ER and Golgi (on the noncytosolic side of the membrane).
Proteins - Glycosylation occurs similarly in glycolipids, modifying proteins and lipids for functional roles.

- The cytosolic environment is reducing, preventing S–S bond formation.

Disul de Bond
- Disul de bonds form on the noncytosolic side, stabilizing protein structures or facilitating
Formation
polypeptide interactions.

This electron micrograph of the surface of a lymphocyte

stained with ruthenium red emphasizes the thick
carbohydrate-rich layer surrounding the cell.
- Most extracellular membrane proteins are glycosylated, forming a carbohydrate coat around the
Carbohydrate cell.
Coating & - Carbohydrates attach to glycoproteins, glycolipids, and proteoglycans.
Glycocalyx - Proteoglycans consist of long polysaccharide chains covalently linked to a protein core.
- Some proteoglycans extend across the membrane or attach via a GPI anchor.
- Protects cells from mechanical and chemical damage.
Function of the
- Prevents unwanted cell–cell interactions by keeping cells at a distance.
Glycocalyx
- Can be visualized using stains (e.g., ruthenium red) or uorescently labeled lectins.
Diversity of - Oligosaccharide chains are highly diverse, with varied branching patterns and covalent linkages.
Oligosaccharide - Even three sugars can form hundreds of di erent trisaccharides.
Chains - The diversity & surface exposure of oligosaccharides enable cell recognition functions.

The carbohydrate layer is made up of the oligosaccharide

side chains of membrane glycolipids and membrane
glycoproteins and the polysaccharide chains on
membrane proteoglycans.
Role in Cell - Oligosaccharides on glycoproteins and glycolipids play key roles in cell recognition.
In addition, adsorbed glycoproteins, and adsorbed
Recognition & - Plasma-membrane-bound lectins bind speci c oligosaccharides, facilitating cell-cell adhesion. proteoglycans (not shown), contribute to the carbohydrate
Adhesion - Important in lymphocyte recirculation & in ammatory responses. layer in many cells. Note that all of the carbohydrate is on
the extracellular surface
of the membrane.
fi
fi
fl
ff
fi
fi
fl
fi
 Solubilization and Puri cation of Membrane Proteins in Detergents
Role of - Membrane proteins require detergents to disrupt hydrophobic
Detergents in interactions and extract them from the lipid bilayer.
Solubilizing - Detergents are small amphiphilic molecules that bind to
Membrane hydrophobic regions of proteins, replacing lipid molecules.
Proteins

Types of - Ionic detergents (e.g., SDS) disrupt membranes strongly,

Detergents denaturing proteins.
- Nonionic detergents (e.g., Triton X-100, Octylglucoside) gently
solubilize proteins without unfolding them.

Critical - At low concentrations, detergents exist as monomers.

Micelle - When detergent concentration exceeds CMC, they form micelles,
Concentration which help solubilize proteins.
(CMC) - The CMC and micelle size vary by detergent and depend on
temperature, pH, and salt concentration.

Detergent- - Detergents bind hydrophobic membrane proteins, forming

Protein detergent-protein complexes that keep them soluble in water. Solubilizing a membrane protein with a mild nonionic detergent. The detergent
Complexes - Some lipids may remain attached, affecting protein stability and disrupts the lipid bilayer and brings the protein into solution as protein–lipid–
detergent complexes. The phospholipids in the membrane are also solubilized by
function. the detergent, as lipid-detergent micelles.

SDS-PAGE & - SDS (sodium dodecyl sulfate) is a strong ionic detergent used for
Protein polyacrylamide-gel electrophoresis (SDS-PAGE).
Puri cation - SDS unfolds proteins, allowing separation based on size rather than
shape.
- Some proteins can renature after SDS removal, regaining their
functionality.

Mild - Mild detergents allow solubilization without denaturation,

Detergents for keeping proteins in their active form.
Functional - These detergents coat exposed hydrophobic regions but do not
Puri cation disrupt the protein’s overall structure.

Reconstitution - If detergent is removed in the presence of phospholipids, proteins

into Liposomes can reincorporate into arti cial liposomes.
- This allows functional reconstitution, making it possible to study
transporters, ion channels, and signaling receptors.

ATP Synthase - Experimental reconstitution demonstrated that ATP synthases use

Functional H+ gradients in mitochondria, chloroplasts, and bacteria to
Reconstitution generate ATP.

The use of mild nonionic detergents for solubilizing, purifying, and reconstituting
functional membrane protein systems.
fi
fi
fi
fi
Membrane Protein Reconstitution in Nanodiscs
- Membrane proteins can be reconstituted into nanodiscs, which are
Nanodiscs for
small membrane patches surrounded by a protein belt.
Membrane Protein
- The belt is derived from high-density lipoproteins (HDL),
Study
stabilizing the membrane fragment and keeping it soluble.
- Maintain proteins in their native membrane-like environment.
- Allow access to both sides of the membrane protein for binding
Advantages of Nanodiscs
studies.
- Useful for ligand-binding experiments and functional assays.
- Nanodiscs allow single-particle electron microscopy, enabling high-
Electron Microscopy resolution structural determination.
Analysis - This technique does not require protein crystallization, which is
often dif cult for membrane proteins.

- Detergents are essential for extracting and purifying membrane

proteins.
Model of a membrane protein reconstituted into a nano-disc.
Detergents in Protein - Advancements in detergent chemistry and expression systems have
When detergent is removed from a solution containing a multipass
Puri cation & greatly increased the number of known membrane protein
membrane protein, lipids, and a protein subunit of the high-density
Crystallization structures.
lipoprotein (HDL), the membrane protein becomes embedded in a
- Despite progress, fewer membrane protein structures are known
small patch of lipid bilayer, which is surrounded by a belt of the
compared to soluble proteins due to puri cation challenges.
HDL protein. In such nano-discs, the hydrophobic edges of the
bilayer patch are shielded by the protein belt, which renders the
assembly water-soluble.

Bacteriorhodopsin – A Light-Driven Proton Pump

- First membrane transport protein whose 3D structure was

determined.
Bacteriorhodopsi
- Serves as a prototype for many multipass transmembrane
n Overview
proteins.
- Structure was con rmed and re ned via X-ray crystallography.

- Composed of 7 transmembrane α-helices, tightly packed in the

membrane.
Structure & - Contains a light-absorbing chromophore (retinal), covalently
Function linked to lysine.
- Activated by light, triggering H+ transfer from inside to outside
the cell.

- Retinal (Vitamin A aldehyde) absorbs light and changes Patches of purple membrane, which contain bacteriorhodopsin in the
archaeon Halobacterium salinarum. (A) These archaea live in saltwater pools,
conformation.
where they are exposed to sunlight. They have evolved a variety of light-
Retinal & Light - Conformational shift in the protein leads to proton (H+) activated proteins, including bacteriorhodopsin, which is a light-activated H+
Activation transport across the membrane. pump in the plasma membrane. (B) The bacteriorhodopsin molecules in the
- In bright light, pumps hundreds of H+ per second, generating a purple membrane patches are tightly packed into two-dimensional crystalline
arrays. (C) Details of the molecular surface visualized by atomic force
proton gradient.
microscopy. With this technique, individual bacteriorhodopsin molecules can
be seen. (D) Outline of the approximate location of the bacteriorhodopsin
monomer and the individual α helices in the image shown in (C).
- The H+ gradient powers ATP synthesis via ATP synthase in the
Energy
plasma membrane.
Generation via H+
- Drives other energy-requiring cellular processes in
Gradient
Halobacterium salinarum.
- Bacteriorhodopsin molecules form a planar 2D crystal in
Purple Membrane specialized purple membrane patches.
of Halobacterium - Their tightly packed structure enabled early electron
microscopy & di raction analysis.
- Speci c lipid-protein interactions stabilize bacteriorhodopsin
Lipid Interactions and aid crystallization.
& Stability - Some membrane proteins only function in the presence of
speci c lipid head groups.
The three-dimensional structure of a bacteriorhodopsin molecule.A)
- Part of a superfamily of structurally similar membrane The polypeptide chain crosses the lipid bilayer seven times as α
proteins with diverse functions. helices. The location of the retinal chromophore (purple) and the
Evolutionary
- Rhodopsin in rod cells and G-protein-coupled receptors probable pathway taken by H+ during the light-activated pumping
Homologs
(GPCRs) share a 7-helix structure but have no sequence cycle are shown. The rst and key step is the passing of an
similarity. H+ from the chromophore to the side chain of aspartic acid 85 that
occurs upon absorption of a photon by the chromophore.
Subsequently, other H+ transfers complete the pumping cycle and
- Channelrhodopsins in algae detect light and function as ion
Channelrhodopsi return the enzyme to its starting state. Color code: glutamic acid
channels.
ns in (orange), aspartic acid (red), arginine (blue). (B) The high-resolution
- Engineered channelrhodopsins allow neurons to be activated
Neuroscience crystal structure of bacteriorhodopsin shows many lipid molecules
by light, revolutionizing neurobiology research.
(yellow with red head groups) that are tightly bound to speci c
places on the surface of the protein.
fi
fi
fi
fi
ff
fi
fi
fi
fi
fi
 Membrane Protein Complexes and Di usion
- Many membrane proteins function as part of multicomponent
complexes.
- First membrane protein complex studied: Bacterial
Membrane
photosynthetic reaction center, analyzed by X-ray di raction.
Proteins in Large
- Large complexes are essential for energy harvesting, proton
Complexes
pumping, electron transport, and signal transduction.
- Example: Photosystem II in cyanobacteria has 19 protein
subunits & 60+ transmembrane helices.

- Membrane proteins do not ip- op across the bilayer but can

rotate (rotational di usion) and move laterally (lateral
Membrane Protein di usion). In this experiment, a mouse and a human cell were fused to create a hybrid
cell, which was then stained with two uorescently labeled antibodies. One
Mobility - Heterokaryon Experiment: Fusion of mouse and human cells antibody (labeled with a green dye) detects mouse plasma membrane
showed mixing of plasma membrane proteins within 30 minutes, proteins, the other antibody (labeled with a red dye) detects human plasma
proving lateral di usion. membrane proteins. When cells were stained immediately after fusion, mouse
and human plasma membrane proteins are still found in the membrane
domains originating from the mouse and human cell, respectively. After a
short time, however, the plasma membrane proteins diffuse over the entire
cell surface and completely intermix.

- Fluorescence Recovery After Photobleaching (FRAP)

measures lateral di usion rates of membrane proteins.
Measuring Protein - Process: A uorescently labeled membrane protein is
Di usion (FRAP bleached with a laser, and the time taken for unbleached
Technique) proteins to di use into the area is recorded.
- Common markers: Fluorophore-labeled antibodies or GFP-
tagged proteins (via recombinant DNA technology).
- FRAP tracks large populations, not individual molecules.
- If a protein remains in a bleached area, it may be immobile or
FRAP Limitations
restricted in a con ned region by cytoskeletal interactions.
& Single-Particle
- Single-particle tracking (SPT): Labels individual proteins with
Tracking
uorescent dyes or gold particles, tracking di usion paths via
video microscopy.

- Membrane protein di usion rates vary widely based on A speci c protein of interest can be expressed as a fusion protein
interactions with other proteins and cellular structures.
Variability in with green uorescent protein (GFP), which is intrinsically
- Some freely di use, while others are restricted by cytoskeletal
Membrane Protein uorescent. The uorescent molecules are bleached in a small area
barriers.
Di usion using a laser beam. The uorescence intensity recovers as the
- Studies suggest cell membranes have viscosity comparable to bleached molecules diffuse away and unbleached molecules diffuse
olive oil. into the irradiated area (shown here in side and top views). The
diffusion coef cient is calculated from a graph of the rate of
recovery: the greater the diffusion coef cient of the membrane
protein, the faster the recovery.

Membrane Protein & Lipid Con nement in Speci c Domains

- Biological membranes were originally thought to be uniform
Membranes as Two- lipid bilayers where proteins move freely.
Dimensional Fluids - However, most cells con ne proteins & lipids to speci c
regions, forming membrane domains.

- Bacteriorhodopsin in Halobacterium: Forms 2D crystalline

Examples of arrays in the purple membrane, where protein molecules are
Restricted Protein xed.
Di usion - ATP synthase in mitochondria: Organizes into long double
rows, limiting di usion.
fl
fl
fi
ff
ff
ff
ff
fi
fl
fi
fl
ff
ff
ff
fl
ff
fi
ff
ff
ff
fl
fi
fl
fl
fl
fi
fi
ff
ff
ff
fi
fi
 - Epithelial cells (gut, kidney) have asymmetric membrane Three domains in the plasma membrane of a guinea pig sperm. (A) A
drawing of a guinea pig sperm. (B–D) In the three pairs of micrographs,
protein distribution.
phase-contrast micrographs are on the left, and the same cell is shown with
- Apical surface: Contains speci c enzymes & transport cell-surface immuno uorescence staining on the right. Di erent monoclonal
Membrane Domains
proteins. antibodies selectively label cell-surface molecules on (B) the anterior head,
in Epithelial Cells (C) the posterior head, and (D) the tail.
- Basal & lateral surfaces: Contain di erent proteins.
- Tight junctions prevent lateral di usion, maintaining domain
separation.
- Nanoscale lipid rafts form specialized signaling and
Lipid Rafts & Protein tra cking domains.
Interaction Domains - These rafts are not de ned by tight junctions but by protein–
protein interactions.
- Sperm cells have distinct plasma membrane regions, each
with speci c proteins.
Membrane Domains - Some proteins di use freely within their domain, but do not
in Sperm Cells cross into other domains.
- The mechanism of con nement ("molecular fence") is
unknown.
- Nerve cells have distinct membrane regions:
- Cell body & dendrites (one domain).
Membrane Protein
- Axon (another domain).
Fencing in Neurons
- A belt of actin laments at the cell-body–axon junction acts
as a barrier to protein di usion.

- Four main ways to restrict membrane protein movement

(Figure 10–36):
Mechanisms of
1. Self-assembly into large aggregates (e.g., ATP synthase).
Protein
2. Anchoring to extracellular matrix proteins.
Immobilization
3. Binding to cytoskeletal elements (actin laments).
4. Interacting with proteins on adjacent cells.

The Cortical Cytoskeleton and Membrane Protein Di usion

- Membrane protein mobility is restricted by anchoring to macromolecular assemblies on both
Role of the Cortical sides of the membrane.
Cytoskeleton - Example: Red blood cells maintain their biconcave shape due to interactions between
plasma membrane proteins and the cytoskeleton.

- The red blood cell cytoskeleton consists primarily of spectrin, a exible lamentous protein
(~100 nm long).
Spectrin-Based
- Function: Maintains membrane integrity, allowing the cell to withstand mechanical stress in
Cytoskeleton in Red
capillaries.
Blood Cells
- Spectrin-de cient mice & humans develop anemia; their red blood cells become spherical
& fragile.

- Most cells have a cortical cytoskeletal network, which is more complex than that of red
Actin-Based Cortical blood cells.
Cytoskeleton in Other - Rich in actin laments, which help anchor the plasma membrane.
Cells - Functions: Supports cell movement, endocytosis, and formation of transient structures
( lopodia, lamellopodia).

- The cortical cytoskeleton forms di usion barriers, restricting protein movement.

Membrane Corralling by
- Corrals (membrane domains) can be permanent (e.g., in sperm cells) or transient(e.g.,
Cytoskeleton
protein di usion controlled by cytoskeletal attachment).
- Membrane protein di usion is studied via high-speed single-particle tracking.
Single-Particle Tracking
- Proteins di use within a corral until thermal motion occasionally allows escape.
& Protein Con nement
- The size of a protein's cytoplasmic domain a ects its ability to move across barriers.
- Corralling increases signal e ciency by concentrating receptor complexes.
Corralling & Signal
- Example: When a cell-surface receptor binds an extracellular ligand, a large protein
Transduction
complex forms, making it harder for the receptor to di use away.
fi
ffi
ff
fi
ff
fi
fi
fi
fi
fl
ff
ff
fi
ff
fi
ffi
fi
ff
ff
ff
fi
ff
ff
ff
fl
ff
fi
Membrane-Bending Proteins and Bilayer Deformation
- Cell membranes adopt various shapes: Flat sheets, tubules, vesicles, fenestrated sheets, cisternae.
Membrane Shape & Dynamic Remodeling - Membrane curvature is dynamic and essential for vesicle budding, cell movement, and division.
- Cytoskeletal forces and membrane-bending proteins work together to shape membranes.
- Control local membrane curvature, in uencing vesicle formation, organelle structure, and endocytosis.
Role of Membrane-Bending Proteins
- Attach to speci c membrane regions as needed to drive membrane deformation.
1. Insertion of hydrophobic domains or lipid anchors:
- Proteins insert hydrophobic regions into one lea et, increasing its area and causing bending.
- Example: Proteins shaping ER tubules work this way (Figure 10-40B).

2. Formation of rigid sca olds:

- Some proteins form structural frameworks that either deform membranes or stabilize existing curvature.
Mechanisms of Membrane Bending - Example: Coat proteins involved in vesicle formation (Figure 10-40C).

3. Lipid clustering to induce curvature:

- Speci c lipids cluster in one lea et, a ecting membrane geometry.
- Phosphoinositides have large head groups, making them wedge-shaped, promoting positive curvature
(Figure 10-40D).
- Phospholipases remove lipid head groups, generating negatively curved regions.
Collaborative Action in Membrane - Multiple membrane-bending proteins often work together to achieve a speci c curvature.
Remodeling - Example: Vesicle budding involves multiple curvature-inducing mechanisms(Chapter 13).
fi
fi
ff
fl
ff
fl
fl
fi