Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Chapter 9 Membranes and Cell Surfaces to accompany Biochemistry, 2/e by Reginald Garrett and Charles Grisham All rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company, 6277 Sea Harbor Drive, Orlando, Florida

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Outline 9.1 Membranes 9.2 Structure of Membrane Proteins 9.3 Membrane and Cell-Surface Polysaccharides 9.4 Glycoproteins 9.5 Proteoglycans

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 9.1 Membranes Structures with many cell functions Barrier to toxic molecules Help accumulate nutrients Carry out energy transduction Facilitate cell motion Assist in reproduction Modulate signal transduction Mediate cell-cell interactions

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Spontaneously formed lipid structures Hydrophobic interactions all! Very few lipids exists as monomers Monolayers arrange lipid tails in the air! Micelles bury the nonpolar tails in the center of a spherical structure Micelles reverse in nonpolar solvents

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Spontaneously formed lipid structures Hydrophobic interactions all! Lipid bilayers can form in several ways –unilamellar vesicles (liposomes) –multilamellar vesicles (Alex Bangham)

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company The Fluid Mosaic Model S. J. Singer and G. L. Nicolson The phospholipid bilayer is a fluid matrix The bilayer is a two-dimensional solvent Lipids and proteins can undergo rotational and lateral movement Two classes of proteins: –peripheral proteins (extrinsic proteins) –integral proteins (intrinsic proteins)

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Motion in the bilayer Lipid chains can bend, tilt and rotate Lipids and proteins can migrate ("diffuse") in the bilayer Frye and Edidin proved this (for proteins), using fluorescent-labelled antibodies Lipid diffusion has been demonstrated by NMR and EPR (electron paramagnetic resonance) and also by fluorescence measurements

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Membranes are Asymmetric Lateral Asymmetry of Proteins: –Proteins can associate and cluster in the plane of the membrane - they are not uniformly distributed in many cases Lateral Asymmetry of Lipids: –Lipids can cluster in the plane of the membrane - they are not uniformly distributed

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Membranes are Asymmetric Transverse asymmetry of proteins –Mark Bretscher showed that N-terminus of glycophorin is extracellular whereas C- terminus is intracellular Transverse asymmetry of lipids –In most cell membranes, the composition of the outer monolayer is quite different from that of the inner monolayer

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Flippases A relatively new discovery! Lipids can be moved from one monolayer to the other by flippase proteins Some flippases operate passively and do not require an energy source Other flippases appear to operate actively and require the energy of hydrolysis of ATP Active flippases can generate membrane asymmetries

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Membrane Phase Transitions The "melting" of membrane lipids Below a certain transition temperature, membrane lipids are rigid and tightly packed Above the transition temperature, lipids are more flexible and mobile The transition temperature is characteristic of the lipids in the membrane Only pure lipid systems give sharp, well- defined transition temperatures

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 9.2 Structure of Membrane Proteins Singer and Nicolson defined two classes Integral (intrinsic) proteins Peripheral (extrinsic) proteins We'll note a new one - lipid-anchored proteins

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Peripheral Proteins Peripheral proteins are not strongly bound to the membrane They can be dissociated with mild detergent treatment or with high salt concentrations

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Integral Membrane Proteins Integral proteins are strongly imbedded in the bilayer They can only be removed from the membrane by denaturing the membrane (organic solvents, or strong detergents) Often transmembrane but not necessarily Glycophorin, bacteriorhodopsin are examples

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Glyophorin A single-transmembrane-segment protein One transmembrane segment with globular domains on either end Transmembrane segment is alpha helical and consists of 19 hydrophobic amino acids Extracellular portion contains oligosaccharides and these constitute the ABO and MN blood group determinants Mark Bretscher showed that glycophorin was a transmembrane protein

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Bacteriorhodopsin A 7-transmembrane-segment (7-TMS) protein Found in purple patches of Halobacterium halobium Consists of 7 transmembrane helical segments with short loops that interconnent the helices Note the symmetry of packing of bR (see Figure 9.15) bR is a light-driven proton pump!

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Lipid-Anchored Proteins A relative new class of membrane proteins Four types have been found: –Amide-linked myristoyl anchors –Thioester-linked fatty acyl anchors –Thioether-linked prenyl anchors –Glycosyl phosphatidylinositol anchors

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Amide-Linked Myristoyl Anchors Always myristic acid Always N-terminal Always a Gly residue that links Examples: cAMP-dependent protein kinase, pp60 src tyrosine kinase, calcineurin B, alpha subunits of G proteins, gag protein of HIV-1

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Thioester-linked Acyl Anchors Broader specificity for lipids - myristate, palmitate, stearate, oleate all found Broader specificity for amino acid links - Cys, Ser, Thr all found Examples: G-protein-coupled receptors, surface glycoproteins of some viruses, transferrin receptor triggers and signals

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Thioether-linked Prenyl Anchors Prenylation refers to linking of "isoprene"- based groups Always Cys of CAAX (C=Cys, A=Aliphatic, X=any residue) Isoprene groups include farnesyl (15-carbon, three double bond) and geranylgeranyl (20- carbon, four double bond) groups Examples: yeast mating factors, p21 ras and nuclear lamins

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Glycosyl Phosphatidylinositol Anchors GPI anchors are more elaborate than others Always attached to a C-terminal residue Ethanolamine link to an oligosaccharide linked in turn to inositol of PI See Figure 9.20 Examples: surface antigens, adhesion molecules, cell surface hydrolases

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Lipid Anchors are Signaling Devices Recent evidence indicates that lipid anchors are quite transient in nature Reversible anchoring and de-anchoring can control (modulate) signalling pathways Similar to phosphorylation/ dephosphorylation, substrate binding/ dissociation, proteolytic cleavage triggers and signals

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Bacterial Cell Walls Composed of 1 or 2 bilayers and peptidoglycan shell Gram-positive: One bilayer and thick peptidoglycan outer shell Gram-negative: Two bilayers with thin peptidoglycan shell in between Gram-positive: pentaglycine bridge connects tetrapeptides Gram-negative: direct amide bond between tetrapeptides

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company More Notes on Cell Walls Note the gamma-carboxy linkage of isoglutamate in the tetrapeptide Peptidoglycan is called murein - from Latin "murus", for wall Gram-negative cells are hairy! Note the lipopolysaccharide "hair" in Figures 9.23 and 9.24

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Cell Surface Polysaccharides A host of important functions! Animal cell surfaces contain an incredible diversity of glycoproteins and proteoglycans These polysaccharide structures regulate cell-cell recognition and interaction The uniqueness of the "information" in these structures is determined by the enzymes that synthesize these polysaccharides

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 9.4 Glycoproteins Many structures and functions! May be N-linked or O-linked N-linked saccharides are attached via the amide nitrogens of asparagine residues O-linked saccharides are attached to hydroxyl groups of serine, threonine or hydroxylysine See structures in Figure 9.26

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company O-linked Saccharides of Glycoproteins Function in many cases is to adopt an extended conformation These extended conformations resemble "bristle brushes" Bristle brush structure extends functional domains up out of the glycocalyx See Figure 9.27

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company N-linked Oligosaccharides Many functions known or suspected Oligosaccharides can alter the chemical and physical properties of proteins Oligosaccharides can stabilize protein conformations and/or protect against proteolysis Cleavage of monosaccharide units from N- linked glycoproteins in blood targets them for degradation in the liver - see pages

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 9.5 Proteoglycans Glycoproteins whose carbohydrates are mostly glycosaminoglycans Components of the cell membrane and glycocalyx Consist of proteins with one or two types of glycosaminoglycan See structures, Figure 9.31

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 9.5 Proteoglycans Example: syndecan - transmembrane protein - inside domain interacts with cytoskeleton, outside domain interacts with fibronectin

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Proteoglycan Functions Modulation of cell growth processes –Binding of growth factor proteins by proteoglycans in the glycocalyx provides a reservoir of growth factors at the cell surface Cushioning in joints –Cartilage matrix proteoglycans absorb large amounts of water. During joint movement, cartilage is compressed, expelling water!

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company