BCH422 !

Membrane Proteins: Structure and Function

G. Privé

29 Sept 2015

Lecture 5: !Lipids, membranes and amphiphiles

http://www.lipidmaps.org!http://www.lipidmaps.org/resources

Chemical structures of lipids Enormous diversity. Some of the major classes:

Fatty acyls (~4000 types) fatty acids (C16:0, C16:1, C18:1, C18:2, C20:3, ...)

!Glycerolipids (~3000 types)

triglycerides !Glycerophospholipids (~8000 types)

phosphatidic acid (PA) phosphatidylcholine (PC) phosphatidylserine (PS) phosphatidylinositol (PI) phosphatidylglycerol (PG) phosphatidylethanolamine (PE)

!Sphingolipids (~4000 types)

sphingomyelin glycosphingolipids !

Sterol Lipids (~2000 types) cholesterol

Amphiphiles !• Lipids, detergents, etc !

Defining characteristic: the molecule has a hydrophobic part and a hydrophilic part. !Results in self-assembly in water due to the hydrophobic effect. !The shape and size of the phobic/philic moieties determine how the amphiphile will self-assemble. !The physical properties of a lipid assembly depends on the lipid composition !In mixed systems (e.g. combinations of different lipids), can have concentrations of specific lipids in specific zones.

glycerol

phosphatidylethanolamine (PE)

phosphatidylcholine (PC)

Glycerophospholipids

phosphatidyserine (PS)

Sphingolipids

Glycosphingolipid!!A cerebroside is a !monoglycosylceramide

an amino alcohol N-acylsphingosine!(amide bond formed with a fatty acid)

(C16:0) Lysophosphatidylethanolamine (lysoPE) (a monoglyceride)

cardiolipin

Not all lipids in membranes have two fatty acyl chains!

!Common fatty acids !14:0 myristic acid 16:0 palmitic acid 16:1 cisΔ9 palmitoleic acid 18:0 stearic acid 18:1 cisΔ9 oleic acid 18:2 cisΔ9,12 linoleic acid 18:3 cisΔ9,12,15 linonenic acid 20:4 cisΔ5,8,11,14 arachidonic acid

!fatty acid content varies by tissue

C18:0!stearic acid!Tm=69°C!common in nature

C18:1 cis∆9!oleic acid!Tm=13°C!common in nature!olive oil is mostly triglycerides !with ~60% of the fatty acids as!oleic acid

C18:1 trans∆9!elaidic acid!Tm = 45°C!does not occur naturally!-> hydrogenated oils!

double bonds in fatty acids are fixed as cis or trans (these do not interconvert).

(C16:0, C16:0, C18:1 cis∆9) Triglyceride

(C18:1 cis∆9, C16:0) Phosphatidylcholine (PC; POPC)

(C16:0) Sphingomyelin (SM)

cholesterol

lipid plasma

membrane

ER lysosome mitochondria myelin E. coli

(inner

membrane)

PC 20 48 23 38 11 0

PE 18 19 13 29 17 74

PS 7 4 <1 0 9 0

PI 3 8 6 3 1 0

PG 0 0 0 0 0 19

cardiolipin <1 <1 5 14 0 3

SM 18 5 23 0 8 0

ceramide 3 <1 <1 <1 20 0

cholesterol 20 6 14 3 28 0

others 14 10 16 13 6

Lipid compositions of some membranes

% by weight of total lipids

Alkane dihedral angle - rotation about single bonds (sp3 carbons - tetrahedral)

gauche-transgauche++60° -60°180°

Gauche+ and gauche- are slightly higher in energy than trans, but the barriers to interconversion are small. Saturated chains prefer the trans conformation, but the % of gauche conformation increases with temperature. !!Note: fatty acid chains are usually drawn with the single bonds in the trans conformation. !Do not confuse this with the cis or trans conformation of a double bond (sp2 carbons)! !

cis/trans double bonds are trigonal planar and do not interconvert.

Newman projections:

Gel phase (Lβ’) !• high trans content in the acyl chains • thicker bilayer • less fluid (“frozen”)

Liquid Crystalline phase (Lα) !• mixed trans/gauche+/gauche- content in acyl chains • thinner bilayer • more fluid • headgroups farther apart • most common phase in biological systems

Phase transitions in lipids - Temperature adds thermal energy!

Tm

The Tm for a mixture of lipids is dependent on the headgroup, the acyl chain length, and the presence/degree of unsaturation in the acyl chains

Gel phase few gauche+ or gauche- dihedral angles

Liquid Crystalline phase less order, more dihedral rotations about the alkyl single bonds

cholesteroldi-oleyl

phosphatidylcholine (DOPC)

18:0 sphingomyelin (SM)

Lipid Dynamics !• Internal:

- acyl chain gauche/trans isomerization - dihedral rotations in headgroup - pseudorotation in the sugars - etc.

!• Spatial (rotation/translation of the lipid)

- a soluble molecule has three rotational and three translational degrees of freedom, but a lipid in an assembly is much more constrained in space - rotations in a bilayer:

on axis rotation (fast) no “flipping”

- translations in a bilayer: in plane (x,y relatively fast) out of plane (z) very restricted

Text

Lipid rafts (lateral asymmetry within the membrane) !Certain glycolipids associate more strongly with each other than with phospholipids. These lipids segregate into “domains” or “rafts”. These domains are also enriched in cholesterol. !Rafts can be isolated as detergent insoluble fractions. These are often enriched in signaling molecules (lipids and proteins). This may be used as a mechanism to cluster or localize certain proteins in a membrane.

sphingolipidsphospholipids

cholesterol

Hydrophobic mismatch: when the TM region does not match width of the bilayer. Results in adjustments of the MP conformation and/or the lipids near the MP

Amphiphiles !• Lipids, detergents, etc !

Defining characteristic: the molecule has a hydrophobic part and a hydrophilic part. !Results in self-assembly due to the hydrophobic effect. !The shape and size of the phobic/philic moieties determines how the amphiphile will self-assemble. !The physical properties of a lipid assembly depends on the lipid composition !In mixed systems (e.g. combinations of different lipids), can have concentrations of specific lipids in specific zones.

SDS

Lauryl dimethyl amine oxide (LDAO)

Octyl glucoside (OG)

Dodecyl maltoside (DDM)

Some detergentsD

enat

urin

gSt

abiliz

ing

curvature towards watercurvature away from water

micelles bilayer-forming Non-bilayer-forming

But can be found in membranes with mixed lipid

composition - introduces strain

Amphiphile self-assembly

detergents,

size of headgroup

size of tail

Fact Fiction

Micelle cross section

Total detergent added (mM)

Con

cent

ratio

n (m

M)

total

micelle

monomer

0

0.2

0.4

0.6

0.8

0 0.2 0.4 0.6 0.8

Critical micelle concentration (cmc)

Detergents self-assemble into micelles

below cmc

above cmcdifferent detergents have different cmc values

- Irregular surface packing with significant amount of trans character - non-uniform distribution of headgroups - many exposed acyl chains - the aggregation number is an ensemble average. There is a distribution of micelle sizes. - cmc reflects the monomer solubility !

Disorder and dynamics in detergent micelles

John Holyoake, Régis Pomès

name formula logP solubility in water cmc

ethanol CH -0.3 miscible

butanol CH +0.8 9% (v/v)

octanol CH +3.1 immiscible

sodium decyl sulfate CH 30 mM

sodium dodecyl sulfate (SDS)

CH 2 mM

sodium tetradecyl sulfate CH 0.9 mM

Solubility, logP and cmc - all due to the hydrophobic effect

long

er c

hain

slo

nger

cha

ins

Membrane Proteins and Detergents

• The hydrophobic surfaces of membrane proteins interact with lipophilic groups !

• Normally stabilized in a lipid bilayer (membranes, liposomes) !

• Detergent micelles should mimic the bilayer environment !

• Ideal detergents should solubilize the protein but have no other effect

Protein Detergent Complex (PDC)

1. Crystallize the protein

2. Collect a series of x-ray diffraction images on the crystal

– The crystal provides a diffraction grating that leads to constructive and destructive interference of the x-ray waves as it passes through the crystal lattice

– The resulting diffraction “spots” contain information about the 3D positions of the atoms (electrons) in the protein

3. The image of the protein computationally reconstructed from the diffraction data.

4. A molecular model is built into the image of the electron density

– The model is the x,y,z positions of all the atoms in the protein (PDB file)

Structure determination by x-ray crystallography in a nutshell

• The protein hydrophobic surface is normally embedded in the lipid bilayer

• A protein in a bilayer cannot normally form a 3D crystal

– a protein must be extracted from the membrane with detergents for crystallization trials

• Solubilized proteins exist as Protein-Detergent Complexes (PDC)

– the detergent belt substitutes for the lipid bilayer

– ideal detergents should solubilize the protein but have no other effect

• The bound detergent can double the MW of the MP

– can have several hundred detergent molecules in a PDC

Membrane Protein Crystallization

Membrane Protein embedded in a Lipid Bilayer

Protein-Detergent Complex (PDC)

From Branden & Tooze Introduction to Protein Structure.

Crystallization is the bottleneck in the determination of membrane protein structures.!

The main problem is that the detergent surface in the PDC is “fuzzy” - flexible and dynamic - and is not well-suited for the formation of strong intermolecular contacts. !

Lattice contacts between PDCs are mostly between the polar parts of the proteins. !

!Result - difficult to crystallize, and crystals are often poorly ordered (low resolution).

Crystal lattice of protein-detergent complexes

Lattice

Think of the consequences of making the protein surface more dynamic as in a PDC

The proteins (here shown as discs) would have a “soft” surface and not favour strong, rigid contacts required for a crystal lattice.

Protein-detergent complex crystals

Protein and detergent co-exist in the lattice - but usually only the protein is well-ordered and produces clear electron density.

BCH422 !

Membrane Proteins: Structure and Function

G. Privé

29 Sept 2015

Lecture 5: !Lipids, membranes and amphiphiles

http://www.lipidmaps.org!http://www.lipidmaps.org/resources

Chemical structures of lipids Enormous diversity. Some of the major classes:

Fatty acyls (~4000 types) fatty acids (C16:0, C16:1, C18:1, C18:2, C20:3, ...)

!Glycerolipids (~3000 types)

triglycerides !Glycerophospholipids (~8000 types)

phosphatidic acid (PA) phosphatidylcholine (PC) phosphatidylserine (PS) phosphatidylinositol (PI) phosphatidylglycerol (PG) phosphatidylethanolamine (PE)

!Sphingolipids (~4000 types)

sphingomyelin glycosphingolipids !

Sterol Lipids (~2000 types) cholesterol

Amphiphiles !• Lipids, detergents, etc !

Defining characteristic: the molecule has a hydrophobic part and a hydrophilic part. !Results in self-assembly in water due to the hydrophobic effect. !The shape and size of the phobic/philic moieties determine how the amphiphile will self-assemble. !The physical properties of a lipid assembly depends on the lipid composition !In mixed systems (e.g. combinations of different lipids), can have concentrations of specific lipids in specific zones.

glycerol

phosphatidylethanolamine (PE)

phosphatidylcholine (PC)

Glycerophospholipids

phosphatidyserine (PS)

Sphingolipids

Glycosphingolipid!!A cerebroside is a !monoglycosylceramide

an amino alcohol N-acylsphingosine!(amide bond formed with a fatty acid)

(C16:0) Lysophosphatidylethanolamine (lysoPE) (a monoglyceride)

cardiolipin

Not all lipids in membranes have two fatty acyl chains!

!Common fatty acids !14:0 myristic acid 16:0 palmitic acid 16:1 cisΔ9 palmitoleic acid 18:0 stearic acid 18:1 cisΔ9 oleic acid 18:2 cisΔ9,12 linoleic acid 18:3 cisΔ9,12,15 linonenic acid 20:4 cisΔ5,8,11,14 arachidonic acid

!fatty acid content varies by tissue

C18:0!stearic acid!Tm=69°C!common in nature

C18:1 cis∆9!oleic acid!Tm=13°C!common in nature!olive oil is mostly triglycerides !with ~60% of the fatty acids as!oleic acid

C18:1 trans∆9!elaidic acid!Tm = 45°C!does not occur naturally!-> hydrogenated oils!

double bonds in fatty acids are fixed as cis or trans (these do not interconvert).

(C16:0, C16:0, C18:1 cis∆9) Triglyceride

(C18:1 cis∆9, C16:0) Phosphatidylcholine (PC; POPC)

(C16:0) Sphingomyelin (SM)

cholesterol

lipid plasma

membrane

ER lysosome mitochondria myelin E. coli

(inner

membrane)

PC 20 48 23 38 11 0

PE 18 19 13 29 17 74

PS 7 4 <1 0 9 0

PI 3 8 6 3 1 0

PG 0 0 0 0 0 19

cardiolipin <1 <1 5 14 0 3

SM 18 5 23 0 8 0

ceramide 3 <1 <1 <1 20 0

cholesterol 20 6 14 3 28 0

others 14 10 16 13 6

Lipid compositions of some membranes

% by weight of total lipids

Alkane dihedral angle - rotation about single bonds (sp3 carbons - tetrahedral)

gauche-transgauche++60° -60°180°

Gauche+ and gauche- are slightly higher in energy than trans, but the barriers to interconversion are small. Saturated chains prefer the trans conformation, but the % of gauche conformation increases with temperature. !!Note: fatty acid chains are usually drawn with the single bonds in the trans conformation. !Do not confuse this with the cis or trans conformation of a double bond (sp2 carbons)! !

cis/trans double bonds are trigonal planar and do not interconvert.

Newman projections:

Gel phase (Lβ’) !• high trans content in the acyl chains • thicker bilayer • less fluid (“frozen”)

Liquid Crystalline phase (Lα) !• mixed trans/gauche+/gauche- content in acyl chains • thinner bilayer • more fluid • headgroups farther apart • most common phase in biological systems

Phase transitions in lipids - Temperature adds thermal energy!

Tm

The Tm for a mixture of lipids is dependent on the headgroup, the acyl chain length, and the presence/degree of unsaturation in the acyl chains

Gel phase few gauche+ or gauche- dihedral angles

Liquid Crystalline phase less order, more dihedral rotations about the alkyl single bonds

cholesteroldi-oleyl

phosphatidylcholine (DOPC)

18:0 sphingomyelin (SM)

Lipid Dynamics !• Internal:

- acyl chain gauche/trans isomerization - dihedral rotations in headgroup - pseudorotation in the sugars - etc.

!• Spatial (rotation/translation of the lipid)

- a soluble molecule has three rotational and three translational degrees of freedom, but a lipid in an assembly is much more constrained in space - rotations in a bilayer:

on axis rotation (fast) no “flipping”

- translations in a bilayer: in plane (x,y relatively fast) out of plane (z) very restricted

Text

Lipid rafts (lateral asymmetry within the membrane) !Certain glycolipids associate more strongly with each other than with phospholipids. These lipids segregate into “domains” or “rafts”. These domains are also enriched in cholesterol. !Rafts can be isolated as detergent insoluble fractions. These are often enriched in signaling molecules (lipids and proteins). This may be used as a mechanism to cluster or localize certain proteins in a membrane.

sphingolipidsphospholipids

cholesterol

Hydrophobic mismatch: when the TM region does not match width of the bilayer. Results in adjustments of the MP conformation and/or the lipids near the MP

Amphiphiles !• Lipids, detergents, etc !

Defining characteristic: the molecule has a hydrophobic part and a hydrophilic part. !Results in self-assembly due to the hydrophobic effect. !The shape and size of the phobic/philic moieties determines how the amphiphile will self-assemble. !The physical properties of a lipid assembly depends on the lipid composition !In mixed systems (e.g. combinations of different lipids), can have concentrations of specific lipids in specific zones.

SDS

Lauryl dimethyl amine oxide (LDAO)

Octyl glucoside (OG)

Dodecyl maltoside (DDM)

Some detergentsD

enat

urin

gSt

abiliz

ing

curvature towards watercurvature away from water

micelles bilayer-forming Non-bilayer-forming

But can be found in membranes with mixed lipid

composition - introduces strain

Amphiphile self-assembly

detergents,

size of headgroup

size of tail

Fact Fiction

Micelle cross section

Total detergent added (mM)

Con

cent

ratio

n (m

M)

total

micelle

monomer

0

0.2

0.4

0.6

0.8

0 0.2 0.4 0.6 0.8

Critical micelle concentration (cmc)

Detergents self-assemble into micelles

below cmc

above cmcdifferent detergents have different cmc values

- Irregular surface packing with significant amount of trans character - non-uniform distribution of headgroups - many exposed acyl chains - the aggregation number is an ensemble average. There is a distribution of micelle sizes. - cmc reflects the monomer solubility !

Disorder and dynamics in detergent micelles

John Holyoake, Régis Pomès

name formula logP solubility in water cmc

ethanol CH -0.3 miscible

butanol CH +0.8 9% (v/v)

octanol CH +3.1 immiscible

sodium decyl sulfate CH 30 mM

sodium dodecyl sulfate (SDS)

CH 2 mM

sodium tetradecyl sulfate CH 0.9 mM

Solubility, logP and cmc - all due to the hydrophobic effect

long

er c

hain

slo

nger

cha

ins

Membrane Proteins and Detergents

• The hydrophobic surfaces of membrane proteins interact with lipophilic groups !

• Normally stabilized in a lipid bilayer (membranes, liposomes) !

• Detergent micelles should mimic the bilayer environment !

• Ideal detergents should solubilize the protein but have no other effect

Protein Detergent Complex (PDC)

1. Crystallize the protein

2. Collect a series of x-ray diffraction images on the crystal

– The crystal provides a diffraction grating that leads to constructive and destructive interference of the x-ray waves as it passes through the crystal lattice

– The resulting diffraction “spots” contain information about the 3D positions of the atoms (electrons) in the protein

3. The image of the protein computationally reconstructed from the diffraction data.

4. A molecular model is built into the image of the electron density

– The model is the x,y,z positions of all the atoms in the protein (PDB file)

Structure determination by x-ray crystallography in a nutshell

• The protein hydrophobic surface is normally embedded in the lipid bilayer

• A protein in a bilayer cannot normally form a 3D crystal

– a protein must be extracted from the membrane with detergents for crystallization trials

• Solubilized proteins exist as Protein-Detergent Complexes (PDC)

– the detergent belt substitutes for the lipid bilayer

– ideal detergents should solubilize the protein but have no other effect

• The bound detergent can double the MW of the MP

– can have several hundred detergent molecules in a PDC

Membrane Protein Crystallization

Membrane Protein embedded in a Lipid Bilayer

Protein-Detergent Complex (PDC)

From Branden & Tooze Introduction to Protein Structure.

Crystallization is the bottleneck in the determination of membrane protein structures.!

The main problem is that the detergent surface in the PDC is “fuzzy” - flexible and dynamic - and is not well-suited for the formation of strong intermolecular contacts. !

Lattice contacts between PDCs are mostly between the polar parts of the proteins. !

!Result - difficult to crystallize, and crystals are often poorly ordered (low resolution).

Crystal lattice of protein-detergent complexes

Lattice

Think of the consequences of making the protein surface more dynamic as in a PDC

The proteins (here shown as discs) would have a “soft” surface and not favour strong, rigid contacts required for a crystal lattice.

Protein-detergent complex crystals

Protein and detergent co-exist in the lattice - but usually only the protein is well-ordered and produces clear electron density.