G.

Priv
29 Sept 2015

BCH422

Membrane Proteins: Structure and Function

Lecture 5:

!
Lipids, membranes and amphiphiles

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

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

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.

Glycerophospholipids
phosphatidylcholine (PC)

glycerol

phosphatidylethanolamine (PE)

phosphatidyserine (PS)

Sphingolipids

an amino alcohol

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

Glycosphingolipid!

A cerebroside is a !
monoglycosylceramide

Not all lipids in membranes have two fatty acyl chains!

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

!
fatty acid content varies by tissue

!
Common fatty acids
!
14:0
16:0
16:1 cis9
18:0
18:1 cis9
18:2 cis9,12
18:3 cis9,12,15
20:4 cis5,8,11,14

myristic acid
palmitic acid
palmitoleic acid
stearic acid
oleic acid
linoleic acid
linonenic acid
arachidonic acid

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

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

C18:1 cis9!
oleic acid!
Tm=13C!
common in nature!
olive oil is mostly triglycerides !
with ~60% of the fatty acids as!
oleic acid

C18:1 trans9!
elaidic acid!
Tm = 45C!
does not occur naturally!
-> hydrogenated oils!

(C16:0) Sphingomyelin (SM)

(C16:0, C16:0, C18:1 cis9) Triglyceride

(C18:1 cis9, C16:0) Phosphatidylcholine (PC; POPC)

cholesterol

Lipid compositions of some membranes

lipid

plasma

ER

lysosome

mitochondria

myelin

membrane

E. coli
(inner
membrane)

PC

20

48

23

38

11

PE

18

19

13

29

17

74

PS

<1

PI

PG

19

cardiolipin

<1

<1

14

SM

18

23

ceramide

<1

<1

<1

20

cholesterol

20

14

28

others

14

10

16

13

% by weight of total lipids

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

Newman projections:

gauche+
+60

trans

gauche-

180

-60

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.

Phase transitions in lipids - Temperature adds thermal energy!

Tm

Gel phase (L)

Liquid Crystalline phase (L)

high trans content in

mixed trans/gauche+/gauche-

the acyl chains

thicker bilayer
less fluid (frozen)

content in acyl chains

thinner bilayer
more fluid
headgroups farther apart
most common phase in
biological systems

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

cholesterol

di-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

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.

Text

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.

phospholipids
sphingolipids
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.

Denaturing

Some detergents

SDS

Lauryl dimethyl amine oxide (LDAO)

Stabilizing

Octyl glucoside (OG)

Dodecyl maltoside (DDM)

Amphiphile self-assembly
curvature away from water

curvature towards water

size of headgroup
size of tail
,
detergents

micelles

bilayer-forming

Non-bilayer-forming

But can be found in

membranes with mixed lipid
composition - introduces strain

Micelle cross section

Fact

Fiction

Detergents self-assemble into micelles

Concentration (mM)

Critical micelle concentration (cmc)

0.8
total

0.6

micelle

0.4

monomer

0.2
0

below cmc

0.2

0.4

0.6

0.8

Total detergent added (mM)

different detergents have different cmc values

above cmc

Disorder and dynamics in detergent micelles

- 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

!
John Holyoake, Rgis Poms

name

formula

logP

solubility in water

longer chains

ethanol

CH

-0.3

miscible

butanol

CH

+0.8

9% (v/v)

octanol

CH

+3.1

immiscible

longer chains

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

cmc

sodium decyl sulfate

CH

30 mM

sodium dodecyl sulfate

(SDS)

CH

2 mM

sodium tetradecyl sulfate

CH

0.9 mM

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)

Structure determination by x-ray crystallography

in a nutshell

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)

Membrane Protein Crystallization

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 embedded

in a Lipid Bilayer

Protein-Detergent Complex (PDC)

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 proteindetergent complexes

From Branden & Tooze Introduction to Protein Structure.

Think of the consequences of making

the protein surface more dynamic as
in a PDC

Lattice

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.

G. Priv
29 Sept 2015

BCH422

Membrane Proteins: Structure and Function

Lecture 5:

!
Lipids, membranes and amphiphiles

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

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

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.

Glycerophospholipids
phosphatidylcholine (PC)

glycerol

phosphatidylethanolamine (PE)

phosphatidyserine (PS)

Sphingolipids

an amino alcohol

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

Glycosphingolipid!

A cerebroside is a !
monoglycosylceramide

Not all lipids in membranes have two fatty acyl chains!

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

!
fatty acid content varies by tissue

!
Common fatty acids
!
14:0
16:0
16:1 cis9
18:0
18:1 cis9
18:2 cis9,12
18:3 cis9,12,15
20:4 cis5,8,11,14

myristic acid
palmitic acid
palmitoleic acid
stearic acid
oleic acid
linoleic acid
linonenic acid
arachidonic acid

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

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

C18:1 cis9!
oleic acid!
Tm=13C!
common in nature!
olive oil is mostly triglycerides !
with ~60% of the fatty acids as!
oleic acid

C18:1 trans9!
elaidic acid!
Tm = 45C!
does not occur naturally!
-> hydrogenated oils!

(C16:0) Sphingomyelin (SM)

cholesterol

(C16:0, C16:0, C18:1 cis9) Triglyceride

(C18:1 cis9, C16:0) Phosphatidylcholine (PC; POPC)

Lipid compositions of some membranes

lipid

plasma

ER

lysosome

mitochondria

myelin

membrane

E. coli
(inner
membrane)

PC

20

48

23

38

11

PE

18

19

13

29

17

74

PS

<1

PI

PG

19

cardiolipin

<1

<1

14

SM

18

23

ceramide

<1

<1

<1

20

cholesterol

20

14

28

others

14

10

16

13

% by weight of total lipids

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

Newman projections:

gauche+
+60

trans
180

gauche-60

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.

Phase transitions in lipids - Temperature adds thermal energy!

Tm

Gel phase (L)

Liquid Crystalline phase (L)

high trans content in

mixed trans/gauche+/gauche-

the acyl chains

thicker bilayer
less fluid (frozen)

content in acyl chains

thinner bilayer
more fluid
headgroups farther apart
most common phase in
biological systems

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

Liquid Crystalline phase

Gel phase
few gauche+ or gauche- dihedral angles

cholesterol

less order, more dihedral rotations about the

alkyl single bonds

di-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

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.

Text

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.

phospholipids
sphingolipids
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.

Denaturing

Some detergents

SDS

Lauryl dimethyl amine oxide (LDAO)

Stabilizing

Octyl glucoside (OG)

Dodecyl maltoside (DDM)

Amphiphile self-assembly
curvature away from water

curvature towards water

size of headgroup
size of tail
,
detergents

micelles

bilayer-forming

Non-bilayer-forming

But can be found in

membranes with mixed lipid
composition - introduces strain

Micelle cross section

Fiction

Fact

Detergents self-assemble into micelles

Concentration (mM)

Critical micelle concentration (cmc)

0.8
total

0.6

micelle

0.4

monomer

0.2
0

below cmc

0.2

0.4

0.6

0.8

Total detergent added (mM)

different detergents have different cmc values

above cmc

Disorder and dynamics in detergent micelles

- 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

!
John Holyoake, Rgis Poms

name

formula

logP

solubility in water

longer chains

ethanol

CH

-0.3

miscible

butanol

CH

+0.8

9% (v/v)

octanol

CH

+3.1

immiscible

longer chains

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

cmc

sodium decyl sulfate

CH

30 mM

sodium dodecyl sulfate

(SDS)

CH

2 mM

sodium tetradecyl sulfate

CH

0.9 mM

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)

Structure determination by x-ray crystallography

in a nutshell

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)

Membrane Protein Crystallization

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 embedded

in a Lipid Bilayer

Protein-Detergent Complex (PDC)

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 proteindetergent complexes

From Branden & Tooze Introduction to Protein Structure.

Think of the consequences of making

the protein surface more dynamic as
in a PDC

Lattice

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.