1. Membrane Proteins
FOB Guided Exploration 9

2. Different membrane proteins are associated with the membranes in different ways.
Membrane proteins can be classified into two broad categories – integral (intrinsic) and peripheral (extrinsic) – based on the nature of the membrane-protein interactions.
Integral membrane proteins have one or more segments that are embedded in the phospholipid bilayer. Most integral protein contain residues with hydrophobic side chains that can interact with fatty acyl groups of the membrane phopholipids thus anchoring the protein to the membrane. Most integral protein span the entire phospholipid bilayer.
Transmembrane proteins are an example of an integral protein.
Peripheral membrane proteins do not interact with the hydrophobic core of the phospholipid bilayer. Instead they are usually bound to the membrane indirectly by interactions with integral membrane proteins or directly by interactions with lipid polar head groups. Peripheral proteins localized to the cytosolic face of the plasma membrane include cytoskeletal proteins spectrin and actin in erythrocytes and the enzyme protein kinase C.
Fig in FOB

3. Membrane Proteins Classes (kind of membrane association):
Transmembrane:
rule: polypeptide chain passes completely through the bilayer
examples:
single membrane-spanning domain: hydrophobic a-helix (e.g., glycophorin)
multiple membrane-spanning domains (e.g., 7-pass transmembrane proteins such as bacteriorhodopsin)
hydropathy plots to detect transmembrane domains
pores: a-helical vs. b-sheet/barrel (porins)
Protein molecules found in the plasma membrane mediate a variety of functions of the membrane, such as transport of molecules across the membrane, catalyzing membrane-associated reactions (ATP synthesis), structural links that that connect the membrane to the cytoskeleton and/or to either the extracellular matrix or an adjacent cell, while others serve as receptors to detect, and transduce chemical signals in the cell’s environment.
Transmembrane proteins extend through the lipid bilayer. They are amphipathic having regions that are hydrophobic and others that are hydrophilic. The hydrophobic regions pass through and interact with the hydrophobic regions on the inside of the bilayer.
The hydrophilic regions are exposed to water on either side of the membrane

4. A transmembrane protein always has a unique orientation in the membrane, reflecting both the assymetrical manner in which it is synthesized in the ER and inserted into the lipid bilayer and the different functions of its cytoplasmic and noncytoplasmic domains. These domains are separated by the membrane-spanning segments of the polypeptide chain, which contact the hydrophobic environment of the lipid bilayer and are composed largely of amino acid residues with nonpolar side chains (green and yellow above). Because the peptides themselves are polar and because water is absent all peptide bonds in the bilayer are driven to form hydrogen bonds with one another. The hydrogen bonding between peptide bonds is maximized if the polypeptide chain forms a regular alpha helix ars it crosses the bilayer, and this is how the great majority of the membrane spanning segments of polypeptide chains are thought to traverse the bilayer.

5. In single pass transmembrane proteins, the polypeptide crosses only once.
In multipass transmembrane proteins, the polypeptide chain crosses multiple times.
An alternative way for the peptide bonds in the lipid bilayer to satisfy their hydrogen-bonding requirements is for multipass transmembrane strands of polypeptide chain to be arranged as a beta sheet in the form of a closed barrel (beta barrel). This form of multipass transmembrane structure is seen in porin proteins.

6. This is a figure of the human erythrocyte protein glycophorin A
This is a figure of the human erythrocyte protein glycophorin A. Using surface labeling – a technique that uses agents that react to protein but cannot penetrate membranes is was discovered that glycophorin A has three domains: (1) a 72-residue externally located N-terminal domain that bears 16 carbohydrate side chains (2) a 19 residue sequence consisting almost entirely of hydrophobic residues, that spans the erythrocyte cell membrane, and (3) a 40-residue cytoplasmic C-terminal domain that has a high proportion of charged and polar residues.
This figure illustrates the fact that integral proteins are amphiphiles. The protein segments located in the nonpolar interior have predominantly hydrophobic surface residues, whereas those portions that extend into the aqueous environment have largely polar residues.
Fig in FOB

7. Bacteriorhodopsin is an example of a multispanning alpha helical transmembrane protein. It is a seven spanning integral protein.
Bacteriorhodopsin is a protein bound in a photosynthetic bacterium. Absorption of light by the retinal group attached to bacteriorhodopsin causes a conformational change in the protein that results in pumping of protons from the cytosol across the extracellular space. The proton gradient are generated across the membrane is used to synthesize ATP.
The alpha helices are labeled here from A to G. The retinal pigment is covalently attached to lysine 216 in helix G.
Fig in MCB

8. Transmembrane proteins are very difficult to crystallize so the 3-D structures of almost all transmembrane proteins are uncertain. DNA cloning and sequencing techniques have revealed that amino acid sequences of large numbers of transmembrane proteins and it is often possible to predict from analysis of the proteins sequence which parts of the polypeptide extend across the lipid bilayer as an alpha helix.
Segments containing about amino acid residues with a high degree of hydrophobicity are long enough to span a membrane as an alph helix and they can often be identified by means of a hydropathy plot.
Basically the free energy needed to transfer successive segment of a polypeptide chain from a nonpolar solvent to water is calculated from the amino acid composition of each using data on model compounds. This calculation is made for segments of a fixed size beginning with each successive amino acid in the chain. The “hydropathy index” is plotted on the Y axis as a function of its location in the chain. A positive value indicates that free energy is required for transfer to water (the segment is hydrophobic) and the value assigned is an index of the amount of energy needed. Peaks in the hydropathy index appear at the positions of hydrophobic segment in the amino acid side chain.

9. 1. Proteins that span a membrane have a characteristic structure in the region of the lipid bilayer. Which, if any, of the three 20-amino acid sequences listed below is the most likely candidate for such a transmembrane segment? Explain the reasons for your choice. A. Ile Thr Leu Ile Tyr Phe Gly Val Met Ala Gly Val Ile Gly Thr Ile Leu Leu Ile Ser B. Ile Thr Pro Ile Tyr Phe Gly Pro Met Ala Gly Val Ile Gly Thr Pro Leu Leu Ile Ser C. Ile Thr Glu Ile Tyr Phe Gly Arg Met Ala Gly Val Ile Gly Thr Asp Leu Leu Ile Ser
A and B
Take note of the charged polar amino acids: Glutamic acid, arginine, ad aspartic acid.

10. The formation of a transmembrane hydrophilic pore by multiple alpha helices. In this example. Five transmembrane alpha helices form a water-filled channel across the lipid bilayer. The hydrophobic side chains (green) on one side of each helix contact the hydrophobic hydrocarbon tails, while the hydrophilic side chains (red) on the opposite side of the helices form the surfaces of a water-filled pore.
Porins are found in the outer membranes of mitochondria and chloroplasts (3-35 MCB, 10-6 FOB).
Several types of porins are found in the outer membrane of gram-negative bacteria such as E. coli. The porins in the outer membrane of the E.coli provide channels for passage of disaccharides, phosphate, and similar molecules. The amino acid sequences are predominantly polar and contain no long hydrophobic segments typical of other integral proteins with alpha-helical membrane spanning domains. In each subunit 16 beta strands form the barrel shaped structure with a pore in the center. Half of the amino acid side groups point in one direction and the other half point in the opposite direction.
Porins have an inside out arrangement the outward facing side groups are hydrophobic and can interact with the fatty acyl groups of the membrane lipids or with other porin monomers. The side groups facing the inside of the porin monomer are predominantly hydrophilic. These line the pore through which small water soluble molecules cross the membrane.

11. These are schematics and x-ray structures of E. coli porins
These are schematics and x-ray structures of E.coli porins. In the figure to the right you can see a pore through each subunit.
Fig in FOB

12. There are six ways in which membrane proteins associate with the lipid bilayer.
Most transmembrane proteins are thought to extend across the bilayer as a single alpha helix (1) or as multiple alpha helices (2), some of these single pass or multi pass proteins have a covalently attached fatty acid side chain inserted in the cytoplasmic monolayer.
Other membrane proteins are attached to the bilayer soley by a covalently attached lipid – either a fatty acid chain or prenyl group – in the cytoplasmic monolayer (4) or, less often, via an oligosaccharide, to a minor phospholipid, phosphatidylinositol in the noncytoplasmic monolayer (5).
Finally many proteins are attached to the membrane only by noncovalent interactions with other membrane proteins (6 and 7)

13. Membrane Proteins Classes (kind of membrane association):
Lipid anchoring (covalent attachment to membrane phospholipid):
For proteins on cytosolic face:
fatty acid:
myristate (C14): amide linkage to amino group of N-terminal glycine
palmitate (C16): thioester linkage to Cys residue
prenylation: e.g. polyisoprenoid (farnesyl or geranylgeranyl group) linked to methylated C-terminal Cys (which was initially four residues from C-terminus, but last three residues are cleaved off, then carboxyl group of Cys is methylated)
For proteins on exoplasmic face:
GPI (glycosylphosphatidylinositol) anchor: amide linkage to C-terminal residue of protein
Peripheral: non-covalent protein-protein interactions w/ integral membrane protein(s). The bulk of the peripheral membrane protein resides entirely on one face of the membrane
Some membrane-associate proteins have one or more covalently attached lipids that anchor the protein to the membrane. The lipid group may mediate protein-protein interactions or modify the structure and activity of the protein to which it is attached.
Lipid linked proteins come in three varieties. Fatty acetylated proteins, prenylated proteins (most common groups are farnesyl and geranyl geranyl), and glycophosphotidylinositol-linked proteins.
Two kinds of fatty acids, myristic acid and palmitic acid, are linked to membrane proteins.
Myristic acid is appended to a protein via an amide linkage to the alpha-amino group of a N-terminal Gly residue. Myristoylation is stable; The fatty acyl group remains attached to the protein throughout its lifetime. These proteins are found in a variety of subcellular compartments including the cytosol, ER, plasma membrane, and the nucleus.
Palmitoylation occurs when the fatty acid palmitic acid is joined in thoieser linkage to a specific cystene residue. Palmitoylated proeins occur almost exclusively on the cytoplasmic face of the plasma membrane where many participate in transmembrane signaling. The palmitoyl group can be removed suggesting that reversible palitolyation may regulate the association of the protein with the membrane abd thereby modulate the signaling process.
Prenylated proteins have covalently attached lipids that are built from isoprene ( a C5 compound) units. The most common isoprenoid groups are the farnesyl (C15) and geranylgeranyl (C20) residues. The most common prenylation site in proteins is the C-terminal tetrapeptide (CXXY) where C is Cys and X is often an aliphatic amino acid group. Residue Y influences the type of prenylation (either farnesylated or geranylgeranylated). In both cases the prenyl group is enzymatically linked to the Cys sulfur atom via a thioester linkage. The XXY tripeptide is then proteolytically excised and the newly exposes terminal carboxyl group is esterified with a methyl group producing a C-terminus with the structure.

14. GPI – linked proteins occur in all eukaryotes but are particularily abundant in some prokaryotic protozoa. These proteins are located only on the exterior surface of the plasma membrane. The core of the GPI group consists of phosphatidylinositol glycosidically linked to a linear tetrasaccharide composed of 3 mannose residues and 1 glucosaminyl residue. The mannose at the nonreducing end of this assembly forms a phosphodiester bond with a phosphoethanolamine residue that is aminde-linked to the proteins C-terminal carboxyl group. The core tetrasaccharide is generally substituted with a variety of sugar residues that vary with the identity of the protein.
Fig in MCB

15. This is an X-ray structure of the photosynthetic reaction center of Rhodopseudomonas viridis. This is the first transmembrane protein to be described in atomic detail.
Fig in FOB

16. Generally, transmembrane proteins can be solubilized only by agents that disrupt hydrophobic associations and destroy the lipid bilayer. The most useful are detergents, which are small amphipathic molecules that tend to form micelles in water.
The hydrophobic part of a detergent molecule is attracted to hydrocarbons and mingles with them readily. The hydrophilic part is strongly attracted to water.
Some detergents are natural products, but most are synthetic molecules developed for cleaning and for the dispersal of mixtures of oil and water. Ionic detergents such as sodium deoxycholate and sodium dodecylsulfate (SDS) , contain a charged group. Nonionic detergents such as triton-X (TTX) and octylglucoside lack charged groups.
At very low concentrations, detergents dissolve in pure water as isolated molecules. As the concentration increases the molecules begin to form micelles. The critical micelle concentration (CMC) at which micelles form is characteristic of each detergent and is a function of the structures of its hydrophobic and hydrophilic parts.
Ionic detergents bind to the exposed hydrophobic regions of membrane proteins as well as to the hydrophobic core of water soluble proteins. Because of their charge they disrupt ionic and hydrogen bonds. At high concentrations sodium dodecylsulfate completely denatures proteins by binding to every side chain.
Nonionic detergents act in different ways at different concentrations. At high concentrations (above the CMC), they solubilize biological membranes by forming mixed micelles of detergent, phospholipid, and integral membrane proteins. At low concentrations these detergents may bind to the hydrophobic regions of most membrane proteins making them soluble in aqueous solution. In this case, although mixed micelles are not formed, the solubilized protein will not aggregate during subseuent purification steps.
Most peripheral proteins are bound to specific integral proteins by ionic or other weak interactions. Generally, they can be removed from the membrane by solutions of high ionic strength (high salt concentrations) which disrupt ionic bonds, or by chemicals that bind divalent cations such as magnesium. Unlike integral proteins most peripheral proteins are soluble is aqueous solution and are not solubilized by nonionic detergents.
Fig in MCB

17. This schematic shows how a mild detergent can disrupt the lipid bilayer and bring the proteins into solution as protein-lipid detergent complexes. The phosphlipids in the membrane are also solubilized by the detergent.

18. Experimental evidence for rapid redistribution of membrane proteins:
Many membrane proteins display rotational and lateral diffusion, but they are much slower than phospholipids (1/10 - 1/100 the rate)
Experimental evidence for rapid redistribution of membrane proteins:
Making heterokaryons – e.g., fusing mouse and human cells; specific cell surface antigens unique to either mouse or human cells were recognized by differentially-labeled antibodies. With time, mixing of cell surface antigens was observed.
Patching and capping – multivalent ligands that recognize specific membrane proteins bind to and cross-link the proteins, which aggregate into clusters (patching); patches are then actively swept to one end of the cell (capping).
Fluorescence recovery after photobleaching (FRAP) – membrane proteins are labeled with a fluorescent molecule; illumination of a small area of the cell surface bleaches out the fluorescence; over time, fluorescent molecules from adjoining unbleached areas are seen to move into the bleached area. Can be used to determine rates of lateral diffusion.
Like membrane lipids, membrane proteins do not flip-flop across the bilayer, but they do rotate about an axis perpendicular to the plane of the bilayer (rotational diffusion). In addition, many proteins are able to move laterally within the membrane (lateral diffusion).
See guided Exploration 9 in FOB
The demonstrated fluidity of artificial lipid bilayers suggests that biological membranes have similar properties.
This idea was proposed using a theory of membrane structure known as the fluid mosaic model. A key element of the model is that integral proteins can diffuse laterally in the lipid matrix unless their movements are restricted by association with other cell components.

19. The first evidence that some plasma membrane proteins are mobile in the plane of the membrane was found in 1970 when mouse cells were artificialy fused with human cells to produce hybrid cells called heterocaryons. In the experiment two differently labeled antibodies were used to distinguish selected mouse and human plasma membrane proteins. At first the mouse and human proteins were confined to their own halves of the newly formed heterocaryon, however, gradually the two sets of proteins diffused and mixed over the entire cell surface within half an hour or so.
Fig in MCB

20. Further evidence for membrane protein mobility was provided by the discovery of the processes called patching and capping.
When ligands, such as antibodies that have more than one binding site (so called multivalent ligands) bind to specific proteins on the surface of cells, the proteins tend to become aggregated, through cross-linking, into large clusters (or patches) indicating that the protein molecules are able to diffuse laterally in the lipid bilayer. Once such clusters have formed on the surface of a cell capable of locomotion, such as a WBC, they are actively moved to one pole of the cell to form a cap.

21. The lateral diffusion rates of membrane proteins can be measured using a technique of flourescence recovery after photobleaching (FRAP). The method usually involves marking the cell surface protein of interest with a specific flourescent ligand, such as a fluorescent antibody. The flourescent ligand is then bleached in a small area by a laser beam, and the time taken for the adjacent membrane proteins carrying unbleached flourescent ligand to diffuse into the bleached area is measured. From such measurements, diffusion coefficients can be calculated for the particular cell-surface protein that was marked. The values of the diffusion coefficients for different membrane proteins in different cells are highly variable, but they are typically about one tenth or one-hundredth of the corresponding values for the phospholipid molecules in the same membrane.
Fig in MCB

22. Factors that limit the diffusion of membrane proteins:
Attachment to the cytoskeleton and/or extracellular matrix.
Assembly of cell-cell adhesion complexes and other cell junctions.
Tight junctions isolate basal-lateral surfaces from apical surfaces in polarized epithelia.
Cells have a way of confining membrane proteins to specific domains in a continuous lipid bilayer.
In epithelial cells, such as those that line the gut or the tubules of the kidney certain plasma membrane enzymes and transport proteins are confined to the apical surface of cells whereas others are confined to the basal and lateral surfaces. This asymmetric distribution of membrane proteins is often essential to the function of the epithelium.
The lipid compositions of the two membranes is also different demonstrating that epithelial cells can prevent the diffusion of lipid as well as protein molecules between the domains. This is true only for lipid molecules in the outer monolayer of the membrane. The separation of both protein and lipid molecules is thought to be maintained at least in part by the barriers set up by a specific type of intercellular junction (tight junction). Clearly, the membrane proteins that form these intercellular junctions cannot be allowed to diffuse laterally in the interacting membranes.
A cell can also create membrane domains without using intercellular junctions. Consider a sperm cell, Spermatozoan are single cells that consist of several structurally and functionally distinct parts covered by a continuous plasma membrane. The plasma membrane consists of at least three distinct domains.
In these two examples, the diffusion of protein and lipid molecules is confined to specialized domains within a continuous plasma membrane. Cells also have more drastic ways of immobilizing certain membrane proteins. One is by restricting the lateral mobility of specific membrane proteins by tethering them to macromolecular assemblies either inside or outside the cell.

23. Attachment to the cytoskeleton and/or extracellular matrix. (A and B)
Factors that limit the diffusion of membrane proteins:
Attachment to the cytoskeleton and/or extracellular matrix. (A and B)
MCB 18-6,7
Assembly of cell-cell adhesion complexes and other cell junctions. ©
Tight junctions isolate basal-lateral surfaces from apical surfaces in polarized epithelia. (D)

24. Here is a figure of the human erythrocyte membrane skeleton.
An RBC must squeeze through narrow blood capillaries without rupturing its membrane. The strength and flexibility of erythrocyte plasma membrane depends on the dense cytoskeletal network that underlies the entire membrane and is attached to it at many points.
You can see that the protein spectrin which forms a dense irregular network that underlies the erythrocyte plasma membrane is the primary component of the erythrocyte cytoskeleton.
There are two dimeric subunits of spectrim, alpha and beta which associate to form head-to-head tetramers. The entire cytoskeleton is arranged in a spoke-and-hub network.
Each spectrin tetramer comprises a spoke, extending from and cross-linking a pair of hubs, called junctional complexes. Each junctional complex is composed of a short actin filament plus adducin, tropomyosin, and tropomodulin. The last two of these proteins strengthen the network by preventing the actin filament from depolarizing.
To ensure that the erythrocyte retains its characteristic shape, the spectrin-actin cytoskeleton is firmly attached to the overlying erythrocyte membrane by two peripheral membrane proteins, each of which binds to a specific integral membrane protein.
Ankyrin connects the center of the spectrin to band 3 protein. Band 4.1 protein, a component of the junctional complex, binds to the integral membrane protein glycophorin. This dual binding ensures that the membrane is connected to both the spokes and the hubs of the spectrin-actin cytoskeleton.
Fig in FOB

25. Fig. 10-16 in FOB This is the Gates and Fences Model
Biological membranes consist of heterogeneous patches of lipids and proteins. The uneven distribution of membrane components may result in part from the influence of the cytoskeleton. It is likely that some integral proteins are firmly attached to elements of the cytoskeleton (A) or are trapped within the spaces defined by those spaces (B). Other membrane proteins may be able to squeeze through the gaps or gates between cytoskeletal components (C), whereas still other proteins can diffuse freely without interacting with the cytoskeleton at all.
Proteins confined by the fences of cytoskeletal elements may form distinct membrane domains which differ from those of surrounding areas.
Fig in FOB

26. Membrane Carbohydrates
Carbohydrates especially abundant on plasma membrane; glycocalyx formed of glycolipids, glycoproteins and proteoglycans.
Sugars can be attached to proteins (almost all) or lipids (1 out of 10).
Asymmetric: only extracellular face of plasma membrane is glycosylated.
Functions:
Protection: important for lubrication and structural integrity of the cell surface.
Important for certain cell-cell recognition events (ex. WBC adhesion to endothelial lining of blood vessels).
Plasma membrane proteins are usually decorated, clothed or hidden by carbohydrates which are present on the surface of all eukaryotic cells. These carbos can exist either as oligosaccharide chains covalently bound to membrane proteins (glycoproteins) and lipids (glycolipids) and as polysaccharide chains of integral membrane proteoglycan molecules.
Proteoglycans, which consist of long polysaccharide chains linked covalently to the protein core, are found mainly outside the cell as part of the extracellular matrix. But integral proteoglycans extend across the lipid bilayer or are attached to the bilayer by an glycosylphosphtidylinositol (GPI) anchor.
The term cell coat or glycocalyx is often used to describe the carbohydrate-rich zone on the cell surface. The glycocalyx usually contains both glycoproteins and proteoglycans that have been secreted into the extracelluar space and then adsorbed onto the cell surface. Many of these adsorbed macromolecules are components of the extracellular matrix.
The oligosaccharide side chains of glycoproteins and glycolipids are enormously diverse in their arrangement of sugars. They usually contain fewer than 15 sugar residues, they are usually branched and can be linked together by a variety of covalent linkages.

27. Fig in FOB