Membranes/membrane proteins

Importance of membranes and membrane proteins General requirements for proteins to reside in biological membranes Different types of membrane proteins -helical proteins Trans-membrane helix interactions

Biological Membranes

Highly selective permeability barriers Amphiphilic organization: Hydrophobic in, hydrophilic out Fluid mosaic model: liquid and asymmetric

Importance of membranes
Compartimentalization: specific chemical environment pH, redox potential, enzymatic composition, ion composition etc.

Higher organization
Compartimentalization: specific chemical environment pH, redox potential, enzymatic composition, ion composition etc. Organization into organelles Lyzosomes contains enzymes for degradation of macromolecules at low pH

Mitochondrion

Membrane structures in organelles

Inside mitochondria and chloroplasts further compartimentalization

Importance of membranes
Chemical insulators: Charge gradient, energy generation Proton gradient, energy generation Ion gradients, signaling

Peter Mitchell's, Chemiosmotic Theory, Nobel prize 1978

Chemiosmotic Theory

Function of membrane proteins

Energy generation Signalling Transport Catalysis Form

Fundamental importance

Fundamental importance

Fundamental importance

genomic composition: 20-30% of the genes code for membrane proteins pdb statistics, 25.000 structures 250 membrane proteins

Medical importance

Cytic fybrosis, chloride transporter Virus entry and maturation, receptors Resistance against cytostatica, multi drug resistance proteins Bacterial infection, adhesins, transporters Certain cancer types Immune system Regulated cell death/apoptosis

Economical importance

marketed small molecule drug targets grouped by class

Fluid mosaic model

Singer and Nicholson, 1972

Integral and Peripheral Membrane Proteins

Integral (yellow): bound by Hydrophobic interactions, can only be removed by disruption of membranes, examples are cytochrome oxidase, GPCRs, channels. Peripheral (blue): Bound by electrostatic and H-bond interactions, i.e. mitochondrial cytochrome c.

Types of Membrane Proteins

-helical and -barrel membrane proteins

Photosystem I

OmpF

It costs energy to break hydrogen bonds in hydrophobic environments

It costs ~ 2 kcal/mol to bring one hydrogen bond from water into alkane. (C=O .H-N). It costs ~6 kcal/mol to bring one hydrated pair of hydrogen bond donor/acceptor into alkane. (C=O H2O; N-H H2O). It thus costs about 4 kcal/mol/peptide bond to maintain unfolded/unsatisfied hydrogen bonds in a membrane: It is impossible for an unfolded protein to reside in a membrane.

Main-chain hydrogen bonds

-helical proteins vs. -barrel proteins

membrane hydrophobic core

The hydrogen-bonding patterns imply that -barrel proteins have to be folded prior to insertion into the membrane, whereas presence of 2ndary structure is sufficient for helical proteins.

Localization of and membrane proteins

-helical proteins
bacterial inner membranes cell membranes ER, golgi complex organelle membranes inner and outer membranes of chloroplast and mitochondria viruses

-barrel protein
outer membranes of Gram-negative bacteria outer membranes of chloroplasts and mitochondria

Polytopic Membrane Proteins: Helical Proteins

Bacteriorhodopsin converts light into electrochemical energy. First structure of membrane protein, done by electron microscopy. 7 Transmembrane helices, almost perpendicular to membrane. Discussed in detail Bioenergetics

Main Chain Hydrogen Bonds in -helices

Main chain hydrogenbond donors and acceptors all participate in hydrogen bonds. Helical membrane proteins are common because it is easy to satisfy hydrogen bond requirements.

Side chains in transmembrane -helices

All side chains point outwards into membrane: They all need to be hydrophobic. helices are readily recognised in sequence by stretches of hydrophobic residues.

Hydrophobic residues in yellow

TM Amino acid composition

TM H. Sapiens (all)

16 14

average occurence

12 10 8 6 4 2 0

Hydrophobic residues are over represented Charged and polar residues are under represented

Am ino acid residue

(adapted from Lui et al. Genome Biol 2002 and with data from Sanger institute)

 helical proteins

Identification of helical membrane proteins Hydrophobicity plots

Arg: -4.500 Asn: -3.500 Asp: -3.500 Cys: 2.500 Gln: -3.500 Gly: -0.400 His: -3.200 Ile: 4.500 Leu: 3.800 Lys: -3.900 Phe: 2.800 Pro: -1.600 Ser: -0.800 Thr: -0.700 Trp: -0.900 Val: 4.200

Kyte and Doolittle hydrophobicity scale -4.5 most polar, 4.5 most hydrophobic
Ala: 1.800 Glu: -3.500 Met: 1.900 Tyr: -1.300

MALEPETVTVSEVVSPEYLDMRRRFWIALMLTIPVVILEMGGHGLKHFISGNGSSWIQLL

- Take average hydrophobicity of 9 residues (a window) assign that to the central residue. - Shift the window by one residue etc.
Kyte, J. and Doolittle, R. 1982. J. Mol. Biol.

Hydropathy plots
MALEPETVTVSEVVSPEYLDMRRRFWIALMLTIPVVILEMGGHGLKHFISGNGSSWIQLL

TM regions
7-8 predicted helices

http://www.expasy.org/tools/protscale.html

Number of TM regions
17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 1 2 3

Predicted number of TM helices in the E. coli genome

Occurence in %

10 11 12 13 14 15 16 17 18

Num ber of helices

(Adapted from Krogh et al, JMB 2001)

TM = Trans Membrane

Number of TM regions
37.5 35 32.5 30 27.5 25 22.5 20 17.5 15 12.5 10 7.5 5 2.5 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Predicted number of TM helices in the E. coli, C. elegans genome

occurence in %

num ber of TM helices

(Adapted from Krogh et al, JMB 2001)

Interface residues

Aromatic residues (Trp, Tyr and Phe) are abundant at the polar/apolar border

Tyrosine hydroxyl point outward

Helix angles
bacteriorhodopsin

Helix angles
SecYEG complex

~0-35o tilt avarage ~ 21o

Van den Burg et al. Nature 2004

The hydrophobic effect

The hydrophobic effect gives soluble proteins their compactness.

aqueous solvent hydrophobic polar ordered solvent

Interaction between helices

What then gives TM proteins their compactness and stability?

aqueous solvent hydrophobic polar

Interaction between helices Polar interactions

Few polar residues in TM regions Those that are often involved in inter-TM hydrogen bonds Rhodopsin

Ubarretxena-Belandia and Engelman, Curr. Op. Struc. Biol. 2001

Interaction between helices Glycophorin A

TM most often form coiled-coils (left-handed, right-handed) Knob into holes packing by hydrophobic residues Close VdW interaction packing Protein-protein packing more efficient that lipid protein packing

LIxxGVxxGVxxT
MacKenzie et al. (1997) Science 276, 131-133.

Helical wheel motif

Helix 3.6 residue per turn Residue n and n+4 point in about same direction Heptad repeat, 7 residues

Interaction between helices GXXXG motif

LIxxGxxxGVxxT in glycophorin A GXXXG (or GG4) very close packing Formation of C -H --- O hydrogen bonds In hydrophobic environment C -H --- O about half strength of ordinary NH --- O
MacKenzie et al. (1997) Science 276, 131-133.

GXXXG motif

Identifying interactions: TOXCAT

TOXCAT system: MBP domainmembrane helix-ToxR domain Oligomerization activates ToxR Oligomeric ToxR controls expression of chloramphenicol-acetyl transferase (CAT) CAT gives resistance against chloramphenicol Giving resistance against
Russ and Engelman PNAS 1999

Identifying interactions: TOXCAT

TOXCAT system: MBP domainmembrane helix-ToxR domain Library of TM helices Transformation Selection on Chloramphenicol plates Higher resistance --> better dimerization low CA

High CA

Other Motifs
SxxSSxxT SxxxSSxxT GxxxxxxG AxxxA

Monotopic membrane proteins

Are embedded in but do not traverse the membrane completely Example: Prostaglandin N-synthase

Prostaglandin H2 synthase-1

Monotopic membrane protein; can only be removed from membrane by addition of detergents. Membrane anchor consists of three helices with hydrophobic side chains. Picot et al. Nature 367, 243 - 249 (1994).

Function Prostaglandin Synthase

Catalyses synthesis of Prostaglandin H2 from arachidonic acid. The fatty acid is a hydrophobic degradation product of lipids. Prostaglandins are signal molecules

Action of aspirin

Enzyme binds substrate through hydrophobic channel. Channel is binding site for aspirin.

Summarized
Importance of membrane proteins Hydrogen bonds satisfied within hydrophobic part of membranes Side-chains of TM a-helix hydrophobic Interactions between TM-helices - Polar interaction side chains - VdW interactions due to close packing - C-H...O hydrogen bond Monotopic membrane protein architecture