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BioSci M160 / MolBio255

Structure-Function Relationships of Integral Membrane Proteins

Hartmut “Hudel” Luecke

Biochemistry, Biophysics &

Computer Science

Email: hudel@uci.eduhttp://urei.bio.uci.edu/

~hudel

Integral Membrane Proteins!!!

http://blanco.biomol.uci.edu/Membrane_Proteins_xtal.html

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)

Def: Membrane

http://en.wiktionary.org/wiki/membranehttp://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 loop3.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 protein3.a polytopic transmembrane β-sheet protein

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.

http://aimediaserver4.com/studiodaily/videoplayer/?src=ai4/harvard/harvard.swf&width=640&height=520

http://multimedia.mcb.harvard.edu/anim_innerlife_hi.html

Movie of (Leukocyte extravasation)

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.

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 c9

C18:2 c9,12

Fatty acids

C18:0

C18:1 c9

What about those healthy omega 3 fatty acids???

C18:3 c9,12,15

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)

Sodium dodecyl sulfate (SDS),an amphipathic molecule

amphi = 2 sidespathic = coming together

Detergents & micelles

11

• 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.

Important detergent properties

• 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).

Some definitions and caveats

Other lipids: cholesterolGreek 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

Micelle Bilayer

The Hydrophobic Effect

Micelle Bilayer

Bilayered mixed micelles or bicelles are magnetically anisotropic, self-assembling model membrane structures comprised of long-chain phospholipids and short-chain detergent molecules.

Bicelle

The Hydrophobic Effect

SUV: small unilamellar vesicle; LUV: large unilamellar vesicle; MLV: multilamellar vesicle; MVV: multivesicular vesicle

Liposome Types

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 Lα-phase dioleoylphosphatidylcholine (DOPC) bilayer. The time-averaged 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.

Lateral diffusion (within leaflet)

Flip-flop (change of leaflet)

Lipid Mobility

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