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BioSci M160 / MolBio255
Structure-Function Relationships of
Integral Membrane Proteins
Hartmut “Hudel” Luecke
Biochemistry, Biophysics &
Computer Science
Email: hudel@uci.edu
http://urei.bio.uci.edu/~hudel
Integral Membrane Proteins!!!
PDB total: 70,303 structures (Jan 2011)
Membrane proteins: 722 (263 unique)
http://blanco.biomol.uci.edu/Membrane_Proteins_xtal.html
PDB total: 105,465 structures (Jan 2015)
Membrane proteins: 1589 (520 unique)
120000
100000
80000
60000
40000
20000
0
1
2
http://blanco.biomol.uci.edu/Membrane_Proteins_xtal.html
Def: Membrane
http://en.wiktionary.org/wiki/membrane
http://en.wikipedia.org/wiki/Cell_membrane
Etymology: from Latin membrana ”skin of body”
Noun: membrane (plural: membranes)
1. A flexible enclosing or separating tissue forming a plane or film and separating two
environments (usually in a plant or animal).
Def: Integral (or transmembrane)
Monotopic membrane proteins are partially inserted into the membrane, but do not cross it:
Schematic representation of the different types of interaction between monotopic membrane proteins and the cell membrane:
1.interaction by an amphipathic α-helix parallel to the membrane plane (in-plane membrane helix)
2.interaction by a hydrophobic loop
3.interaction by a covalently bound membrane lipid (lipidation)
4.electrostatic or ionic interactions with membrane lipids (e.g. through a calcium ion)
en.wikipedia.org/wiki/Integral_monotopic_protein
Def: Integral (or transmembrane)
Polytopic (integral) membrane proteins traverse the bilayer completely one or more times:
Schematic representation of transmembrane proteins:
1.a single transmembrane α-helix (bitopic membrane protein)
2.a polytopic transmembrane α-helical protein
3.a polytopic transmembrane β-sheet protein
Movie of (Leukocyte extravasation)
http://aimediaserver4.com/studiodaily/videoplayer/?src=ai4/harvard/harvard.swf&width=640&height=520
http://multimedia.mcb.harvard.edu/anim_innerlife_hi.html
Cell-cell recognition, actin and microtubule assembly and disassembly, kinesin-mediated vesicle transport, protein synthesis on
ribosomes, ER processing, vesicle fusion, etc.
Some issues: accuracy in temporal and spatial scales, overwhelming kinetic crowdedness of a chemical soup, alternative
models for kinesin, etc.
From another animation: An animated model for processive motility by conventional kinesin. The two heads of the kinesin dimer work in a coordinated manner to move
processively along the microtubule. The catalytic core is bound to a tubulin heterodimer along a microtubule protofilament (the cylindrical microtubule is composed of
13 protofilament tracks). In solution, both kinesin heads contain ADP in the active site (ADP release is rate-limiting in the absence of microtubules). The chaotic motion
of the kinesin molecule reflects Brownian motion. One kinesin head makes an initial weak binding interaction with the microtubule and then rearranges to engage in a
tight binding interaction. Only one kinesin head can readily make this tight interaction with the microtubule, due to restraints imposed by the coiled-coil and pre-stroke
conformation of the neck linker in the bound head. Microtubule binding releases ADP from the attached head. ATP then rapidly enters the empty nucleotide-binding site,
which triggers the neck linker to zipper onto the catalytic core. This action throws the detached head forward and allows it to reach the next tubulin binding site, thereby
creating a 2-head-bound intermediate in which the neck linkers in the trailing and leading heads are pointing forward (post-stroke) and backwards (pre-stroke)
respectively. The trailing head hydrolyzes the ATP, and reverts to a weak microtubule binding state and releases phosphate. Phosphate release also causes the
unzippering of the neck linker. The exact timing of the strong-to-weak microtubule binding transition and the phosphate release step are not well defined from current
experimental data. During the time when the trailing head executes the previously described actions, the leading head releases ADP, binds ATP, and zippers its neck
linker onto the catalytic core. This neck linker motion throws the trailing head forward by 160 Å to the vicinity of new tubulin binding site. After a random diffusional
search, the new lead head docks tightly onto the binding site, which completes the 80 Å step of the motor.
Danielli-Davson model
(1930s and 1940s)
Robertson model
(1950s)
Lenard-Singer model (1960s)
Singer-Nicolson “fluid mosaic” model
(1972)
Myelin, which insulates nerve fibers, contains
18% protein and 76% lipid
Mitochondrial inner membranes contain
76% protein and 24% lipid
Fatty acids
C16:0
C18:0
C18:1 c9
C18:2 c9,12
Fatty acids
C18:0
C18:1 c9
What about those healthy omega 3 fatty acids???
C18:3 c9,12,15
Chemists count from the carbonyl carbon (blue numbering), physiologists count from the
tail (ω) carbon (red numbering)
Glycerides
Phospholipids
-
3
Phospholipids
Detergents
Sodium Dodecyl Sulfate (SDS)
Detergents & micelles
O
H3C (CH2)11
4
S
O
- Na +
O
Sodium dodecyl sulfate (SDS),
an amphipathic molecule
amphi = 2 sides
pathic = coming together
Important detergent properties
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Chemical nature and size of hydrophobic tail
Chemical nature and size of head group
Critical micelle concentration (CMC) and aggregation number.
Effect of solution variables (e.g. salt concentration, temperature, etc.) on CMC and
aggregation number
Solubilizing power - The HLB number is a useful guide
Polyionic detergent micelles can precipitate oppositely charged polyions (e.g. CTAB
precipitation of nucleic acids)
Detergent head groups dictate surface interactions. Headgroups of cationic detergents bind to
anionic glass surfaces leaving the surface "greasy". For similar reasons nonionic detergents
tend to make plastics "greasy".
Binding - Don't forget the influence of protein and lipid binding on free detergent
concentration. Proteins bind twice their weight of SDS and lipids can "consume" 10x their
weight of SDS.
Purity - Most commercial detergents are mixtures. The detergent can be a minor component.
CAUTION - 99% pure usually means 99% detergent which can still be an ill-defined
mixture. Polyoxyethylene detergents often contain iron and peroxides which can inactivate
enzymes. Two batches of a single commercial detergent can differ more than two different,
but similar detergents.
Some definitions and caveats
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Amphiphile or surfactant: a molecule with distinct hydrophilic and hydrophobic surfaces. Any substance which causes a solution to foam is
likely an amphiphile.
Detergent: an amphiphile which disrupts membranes
Micelle: a detergent aggregate
Critical micelle concentration (cmc): concentration above which detergent forms micelles. Above the cmc detergent monomer
concentration is independent of total detergent concentration (at least for "discrete" amphiphiles). For ionic detergents cmc depends on
counterion identity and salt concentration.
Critical micelle temperature (Krafft point): temperature below which micelles are insoluble. The Krafft point of potassium dodecyl sulfate
is notorious for being above room temperature.
Micelle structure: is size and concentration independent for detergents with distinct hydrophilic and hydrophobic domains (e.g. SDS). For
detergents (e.g. bile salt) with less defined hydrophilic and hydrophobic domains, micelle structure varies with and size increases with
detergent concentration.
Aggregation number: number of detergent monomers in a micelle. Detergents which form large micelles (e.g. Triton X-100) are hard to
remove from proteins. Detergents which form small micelles (e.g. deoxycholate) are easier to get rid of.
Cloud point: temperature above nonionic detergents separate into two phases
Head group types: anionic, cationic, nonionic, zwitterionic
HLB number (hydrophile-lyophile balance): an empirical measure of emulsifying power (devised by cosmetic chemists). The HLB number
is the sum of plus numbers assigned for hydrophilic groups and minus numbers assigned for hydrophobic groups. Detergents with HLBs
below 10 are water insoluble. Membranes are commonly solubilized with detergents of HLBs about 15. McCutcheon's contains a table of
HLB values. SDS, with an HLB number of 40, is the best solubilizing agent among common detergents.
Detergent binding: Detergents bind strongly to lipids (and proteins). Unless detergent is in considerable excess, ratio of detergent to lipid
(or protein) is more important than absolute detergent concentration. If, for example residual phospholipid is essential to enzyme activity,
then activity will be especially sensitive to detergent protein ratio. Most proteins do not bind nonionic detergents.
Dialysis of detergents: Micelles dialyze slowly. To dialyze choose a detergent with a high cmc. Incomplete dialysis invalidated early studies
of SDS binding to proteins.
Detergent "matching”: detergents with similar structures and similar HLB numbers are likely interchangeable.
UV absorbance: Where UV absorbance can interfere, choose nonionic detergents with alkyl groups (e.g Triton DF series); avoid those with
alkylphenoxy groups (e.g. Triton X series).
Other lipids: cholesterol
Greek chole (bile) and stereos (solid) with the chemical suffix -ol for an alcohol
Other lipids: cholesterol
Other lipids: cholesterol
About 20-25% of the total daily production (~1 g per day) occurs in the liver;
other sites of high synthesis rates include the intestines, adrenal glands and
reproductive organs.
For a person of about 150 pounds (68 kg)
- the total body content of cholesterol is about 35 g
- the daily internal production is about 1 g
- the typical daily dietary intake is 200 to 300 mg (in the US of A)
Konrad Bloch and Feodor Lynen shared the Nobel Prize in Physiology and Medicine in 1964 for their
discoveries concerning the mechanism and regulation of the cholesterol and fatty acid metabolism. A
large part of this mechanism was subsequently clarified by Michael Brown and Joseph Goldstein in
the 1970s and they received the Nobel Prize in Physiology and Medicine for their work in 1985.
Other lipids: glycolipids
The Hydrophobic Effect
Micelle
Bilayer
The Hydrophobic Effect
Micelle
Bicelle
Bilayer
Bilayered mixed micelles or bicelles are magnetically anisotropic, selfassembling model membrane structures comprised of long-chain
phospholipids and short-chain detergent molecules.
Liposome Types
SUV: small unilamellar vesicle; LUV: large unilamellar vesicle;
MLV: multilamellar vesicle; MVV: multivesicular vesicle
MD Simulation of bilayer
http://blanco.biomol.uci.edu/Bilayer_Struc.html
251 MB version:
http://blanco.biomol.uci.edu/Bilayer_Struc.htmlhttp://blanco.biomol.uci.edu/download/movies/dopc/DOPC_Sim_15Dec08.mov
A representation of the liquid-crystallographic structure of an Lαphase dioleoylphosphatidylcholine (DOPC) bilayer. The timeaveraged transbilayer distributions of the quasi-molecular groups
shown can be thought of as probability or number densities.
"polarity profile" of a DOPC bilayer derived from the structural
image of the previous figure by White and Wimley.
Lipid Mobility
Lateral diffusion (within leaflet)
Flip-flop (change of leaflet)
Types of Membranes
 Plasma membrane
 Nuclear envelope (outer & inner membrane)
 Endoplasmatic reticulum (ER) membrane
 Golgi membrane
 Mitochondrial membrane (outer & inner membrane)
 Vesicles
 Chloroplast
Human Genome
 ~3,000,000,000 bases
 About 1% protein coding, so ~30,000,000 coding bases
 ~10,000,000 amino acids
 Only ~20,000 proteins of 500 amino acids!!!
 Of these between 20% and 25% (or ~5,000) are membrane
proteins