BT631-22-Membrane_proteins

The second large class of proteins distinct from globular proteins are the membrane proteins.

It is estimated that 20-30% of all genes in most genomes encode for membrane proteins.

They are also the target of over 50% of all modern medicinal drugs.

Introduction

The first membrane protein to be

sequenced was Glycophorin.

The first membrane protein structure to be solved was of the bacterial photosynthetic reaction

center from Rhodopseudomonas viridis by Hartmut Michel, Johann Deisenhofer and Robert

Huber for which they shared the Nobel Prize in Chemistry for the year 1988.

Membrane structure and proteins

The exterior membranes

contain many pumps,

channels, receptors and

enzymes and the protein

content of is typically 50%.

Energy-transduction membranes such as the

internal membranes of mitochondria and

chloroplasts have the highest content of protein,

typically 75%.

Genomes and membrane proteins

Wallin & Heijne, 1998, Protein Science , 7, 1029-1038.

The survey of the Human Transmembrane Proteome (UCSF)

33,610 protein sequences in the human genome

29,375 unique protein sequences (no alternative splicing).

7,299 protein sequences are predicted to have at least one transmembrane helix (25%).

3,838 protein sequences predicted to have at least two transmembrane helices (13%).

3,418 unique sequences after removing all residues before and after the first and last

predicted TMH residue.

2,926 unique sequences after clustering at 98% sequence identity.

Distribution of predicted transmembrane helices

3D Structure

As of January 2013 less than 0.1% of protein structures determined were membrane proteins

despite being 20-30% of the total proteome.

RCSB statistics of Membrane structures

Membrane 6727

Membrane part 4378

Membrane-enclosed lumen 1562

Channels/Pores 858

Electrochemical Potential-driven transporters 159

Primary active transporters 735

Group translocators 41

Transmembrane electron carriers 39

Accessory factors involved in transport 224

incompletely characterized transport system 43

Function

Membrane proteins perform a variety of functions vital to the survival of organisms:

TransportSignal transductionEnzymatic activity

Cell-cell recognitionIntercellular joining Attachment to the cytoskeleton

and extra--cellular matrix (ECM)

It is possible to identify membrane proteins by the distribution of residues with hydrophobic

side chains throughout the primary sequence.

Membrane protein prediction

Early prediction of TM segments for helical IMPs generally used the following four-step

procedure

1. Derive propensity scale, a set of 20 numbers corresponding to properties or statistics of the

20 amino acids when found in TM regions.

2. Generate a plot of propensity values along the query sequence.

3. Smooth the plot by taking the average propensity value in a window of N residues and

plot the average at the center of the window (i.e. a sliding window average).

4. Identify TM stretches on the smoothed plot using some propensity threshold.

Amino acid Parameter Amino acid Parameter Amino acid Parameter

Ala 1.80 Gly -0.40 Pro -1.60

Arg -4.50 His -3.20 Ser -0.80

Asn -3.50 Ile 4.50 Thr -0.70

Asp -3.50 Leu 3.80 Tyr -0.90

Cys 2.50 Lys -3.90 Trp -1.30

Gln -3.50 Met 1.90 Val 4.20

Glu -3.50 Phe 2.80

Kyte and Doolittle scheme of ranking hydrophobicity of side chains

Hydropathy plot

Hydropathy plots reflect the preference of amino side chains for polar and non-polar

environments. The hydropathy values reflect measurements of the free energy of transfer of an

amino acid from non-polar to polar solvents.

Modern approaches based on Hidden Markov Models (HMMs) or Neural networks (NNs) are

related to sliding-window hydropathy plot methods.

Membrane Protein Topology

Integral membrane proteins

They can be classified according to their relationship with the bilayer:

IMPs are permanently attached to the membrane and can be separated from the biological

membranes only using detergents, nonpolar solvents or sometimes denaturing agents.

2. Integral polytopic IMPs span across the

membrane at least once. They have one of

two tertiary structures, α bundle and β

barrel (which are found only in outer

membranes of Gram-negative bacteria,

lipid-rich cell walls of a few Gram-positive

bacteria and outer membranes of

mitochondria and chloroplasts).

1. Integral monotopic IMPs are attached to

only one side of the membrane and do not

span the whole way across.

PMPs are temporarily attached either to the lipid bilayer or to integral proteins by a

combination of hydrophobic, electrostatic and/or other non-covalent interactions. Peripheral

proteins dissociate following treatment with a polar reagent, such as a solution with an

elevated pH or high salt concentrations.

Peripheral membrane proteins

PH domain of phospholipase C δ 1

These are located outside the lipid bilayer, on either the extracellular or cytoplasmic surface,

but are covalently linked to a lipid molecule that is situated within the bilayer.

Lipid-anchored proteins

Folding of Membrane Proteins

bovine rhodopsin human mitochondrial voltage-dependent

anion channel

α-helical bundles are structurally and functionally more versatile, serving as receptors,

channels, transporters, electron transporters, and redox facilitators.

β-barrels, made up of β-strands, are found in Gram-negative bacterial outer membranes as

well as in mitochondrial and chloroplast membranes, and these structures function as channels

or transporters for nutrients, proteins, hydrophobic toxic substances, and other molecules.

How do their general structural features compare with those of soluble proteins?

It is almost same. The interior amino acids are found to be almost exclusively nonpolar and

packed just as tightly as those of soluble proteins, as suggested by measurements of the partial

specific volume of bacteriorhodopsin.

Because of the length and the highly nonpolar character of TM helices, hydropathy plots have

proven to be extraordinarily useful and remarkably accurate for predicting the topology of α-

helical MPs.

However, the amino acids of these outer surfaces are more hydrophobic.

The average lengths of the traversing secondary structure elements are greater than for soluble

proteins so that the 30 Å thick bilayer core can be spanned (α-helices are generally longer than

20 amino acids and β-strands longer than 10 amino acids).

Estimating the Molecular Weight of Membrane Proteins

SDS – PAGE is generally used to measure the molecular weight of soluble proteins.

In contrast to the relatively unstructured SDS-induced unfolded states of most water-soluble

proteins, unfolded states of membrane proteins contain a significant percentage of secondary

structure.

It has been observed that native (e.g., glycophorin A) or irreversible (e.g., GPCRs)

oligomerization may occur in SDS micelles.

Also, if not boiled prior to electrophoresis, OmpA migrates to different positions on SDS–

PAGE depending on the compactness of its structure (e.g. native OmpA migrates with an

apparent molecular weight of around 30 kDa, whereas completely unfolded OmpA migrates

as an around 35-kDa protein).

Thus, in some cases, differential binding of SDS to membrane proteins during electrophoresis

has been suggested to cause deviations up to 50% in their apparent molecular weights.

Amino Acid Composition

Leu, Ile and Phe residues are the mostly abundant in the acyl chain areas of membrane lipids.

Lys and Arg residues, with their long and flexible side-chains, are found at the lipid/water

interface region facing cytoplasm (Positive-inside rule). They neutralize their positive charge

by interacting with the negatively charged phosphate groups of phospholipid.

Trp and Tyr residues in the interface and polar residues in the aqueous zone. Trp and Tyr

residues have such a marked tendency to locate in the interfacial area that most membrane

proteins have what is known as an “ aromatic belt. ”

Membrane proteins in Drug Discovery

The G protein-coupled receptors (GPCRs) are the largest, most versatile, group of membrane

receptors and also the most pharmaceutically important, accounting for over 50% of all

human drug targets and acting as therapeutic targets for a wide range of disease conditions

including cancer, cardiovascular, metabolic, CNS and inflammatory diseases.

Ion channels represent another group of important membrane protein drug targets and account

for the activity of 10% of the currently marketed drugs.

Drews, 2000, Science, 287, 1960-1964.

The European Membrane Protein Consortium (http://wwww.e-mep.org)

Japan Biological Information Research Center, Tokyo (http://www.jbic.or.jp/bio/english/)

Centers for Innovation in Membrane Protein Production, UCSF, Scripps ()

Membrane proteins of known structures

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

Transport DB (http://www.membranetransport.org)