BCH 4053 Biochemistry IFall 2001
Dr. Michael Blaber
Membranes, Structure of Membrane Proteins
All cells have a cytoplasmic membrane ("plasma membrane"). One major function of the plasma membrane is to separate the cytoplasm from the outside environment. Other functions related to the plasma membrane include:
- Exclusion or removal of toxic molecules and ions
- Import of cell nutrients
- Energy transduction (typically from surroundings to system)
- Cell locomotion
- Signal transduction
- Cell-cell communication
Membrane environments may be necessary for certain chemical processes essential for life. Thus, the membrane is not only a physical envelope, but a specialized environment for biochemical processes.
Spontaneously formed lipid structures
Monolayers and micelles
Fatty acids spontaneously form various structures in aqueous solution. These are formed because of the hydrophobic effect (i.e. entropy considerations of solvent) that promote the formation of structures that remove the hydrophobic tail of the fatty acid from solvent accessibility.
Monolayer. Fatty acids added to water can form a monolayer on the surface, with the polar head group hydrogen bonding to solvent, and the hydrocarbon tail pointing outwards, away from solvent
- Micelles. Monolayers form with dilute solutions of fatty acids. At higher concentrations (i.e. at the critical micelle concentration, or CMC) fatty acids will associate to form micelle structures. This concentration is typically 0.1 - 10mM.
- Bilayers. Many lipid type molecules energetically prefer to form bilayer structures. You can see that the packing of the hydrocarbon portion of a fatty acid within a micelle can be problematic at the center. For many lipids, optimum van der Waals interactions can be more readily achieve in a side-by-side arrangement found in a bilayer. Bilayers can be quite extensive in area.
- Vesicles. Athough bilayers can be quite extensive, you can see that one problem is that hydrophobic groups are exposed to solvent at the "ends" of the bilayer. This is not energetically favorable, and the bilayer can achieve a lower energy configuration if it wraps around upon itself to form a continuous surface. This results in a physical enclosure termed a unilamellar vesicle. Note that the interior is now a separate and isolated environment from the outer aqueous environment. Such unilamellar vesicles are also called Liposomes.
Several unilamellar vesicles may be located within each other, like the layers of an onion (or those Matrioshka Russian stacking dolls). This organization is called a Multilamellar Vesicle. Note that each compartment can contain a different aqueous environment. (In the following picture the lipid bilayer is represented as a double line)
The bilayer of vesicles has polar surfaces and a hydrophobic middle, and is a barrier to the passage of polar molecules and charged ions into and out of the inner compartment. However, the hydrophobic middle is an excellent environment for non-polar molecules.
The Fluid Mosaic Model
Proposed by Singer and Nicolson in 1972. The model describes the lipid bilayer of vesicles as a dynamic, liquid-like environment that allows the free motion of non-polar molecules throughout its structure. The model also characterizes the lipid bilayer as a complex mixture of both phospholipids and proteins. Both the phospholipids and the proteins may be further conjugated with other groups, such as carbohydrates.
Proteins associated with the lipid bilayer are of two general types:
Peripheral or Extrinsic membrane proteins. These proteins are polar proteins and associate with the polar head groups of the bilayer surface via hydrogen bonds and ionic interactions. They do not penetrate the hydrophobic region of the bilayer, and can be dissociated from the surface under buffer conditions that disrupt ionic interactions (e.g. pH, and high salt)
Integral or Intrinsic membrane proteins. Contain hydrophobic regions or surfaces that penetrate within the hydrophobic region of the lipid bilayer. Integral membrane proteins may extend partly into the hydophobic region, or may span the entire bilayer and have polar regions that face the aqueous environment on both the inside and outside surfaces. Because of their penetration into the hydrophobic region of the bilayer, integral proteins are not readily separated from the bilayer, and require the addition of detergents to remove and solubilize them.
- The lipid bilayer is about 5nm thick (50Å). Thus, an intrinsic membrane protein that spans the membrane has a length on the order of 5nm
- The hydrocarbon tails of the lipid groups may adopt various conformations and may not be exactly perpendicular to the lipid surface. A high degree of mobility is assumed for the hydrocarbon tail of the lipid groups
- Intrinsic membrane proteins have the freedom to move transversely through the lipid bilayer. They can traverse the surface bilayer of an average size eukaryotic cell within minutes. However, their freedom to flip their inside vs. outside orientation is much more limited. Flipping their orientation requires a polar region of the protein to pass through the hydrophobic region of the bilayer. Flipping of integral membrane proteins occurs on a timescale of days.
Membranes are asymmetric structures
Lipid bilayers are typically heterogenous in their constituent lipids. Some lipids may have charged head groups, others may have polar head groups. The different lipid groups may be randomly distributed, or like-molecules may cluster together to produce patches within the bilayer.
- Metal ions, particularly Ca2+, can cause ionic lipids (such as phosphatidyl serine - which has a carboxyl group) to cluster together. This is known as a lateral phase separation of the phosphatidyl serine within the lipid bilayer (lateral because it occurs in the plane of the bilayer).
- Intrinsic proteins can also interact with either the hydrocarbon or head group of specific phospholipids in the bilayer, and cause them to cluster (leading to another lateral phase separation)
- Although the fluid mosaic model describes an environment in the bilayer through which integral proteins can feely move, they also can sometimes exhibit lateral phase separation. This may be due to specific interactions leading to large organized molecular assemblies with the bilayer. Alternatively, some integral membrane proteins interact with molecules within the cell that cause an assembly or concentration in one region of the bilayer.
In addition to lateral asymmetry within the plane of the bilayer, membranes also exhibit transverse asymmetry (i.e. dissimilar properties on the inside versus the outside facing side of the bilayer.
- Intrinsic membrane proteins can exhibit a clear transverse asymmetry. For example, receptor molecules designed to bind signalling molecules in the extracellular environment will be oriented in the bilayer so as to have these binding regions on the side facing the extracellular environment. As we have seen, "flipping" an intrinsic protein requires that a polar or charged region be able to pass through the non-polar region of the bilayer (which would require the input of energy to accomplish)
- Phospholipids also exhibit transverse asymmetry. Glycolipids are important molecules in cell-cell recognition, and must be located on the outer surface of the bilayer to perform this function. Since some phospholipids are charged and others polar, their distribution can affect the charge potential across a lipid bilayer. Such a potential may be critical for cell function, and so a transverse asymmetry of such phospholipids must be established and maintained. Lipid bilayers are synthesized in a transversely asymmetric process, and specific lipid transfer proteins contribute to the maintenance of needed transverse asymmetry in membrane bilayers.
Membrane Phase Transitions
Lipid bilayers can undergo a temperature-dependent phase transition, somewhat like a solid to liquid phase transition.
- At cold temperatures, neighboring phospholipids are closely packed, with trans- carbon-carbon bonds in the hydrocarbon tails. As a consequence there is increased van der Waals contacts between phospholipids and reduced mobility. The phase is gel-like.
- At higher temperatures, there will be a characteristic temperature, the melting temperature (or Tm), where there is a transition to another phase that is a much more mobile, liquid-like phase. This phase transition is a sharp transition and indicates a cooperative transition (all the molecules undergo the phase change at the melting temperature, and the process is therefore cooperative)
- The more liquid like phase is called a "liquid crystal" phase. It is characterized by less well-packed phospholipids, subsequently a larger surface area and thinner bilayer than in the lower temperature "gel" phase. The characteristics of increasing surface area and thinner bilayer are a consequence of the disruption of the packing of the hydrocarbon tails (heat energy disrupts the van der Waals interactions between them)
Features of this phase transition:
It is endothermic - heat is absorbed as it goes through this phase transition
For each type of phospholipid bilayer, there is a characteristic Tm (melting temperature). It increases with chain length (the hydrophobic interactions are greater with longer chain length). Unsaturated bonds can lower the Tm (cis-bonds prevent close-packing and disrupt van der Waals contacts of neighboring lipids)
It is cooperative (the transition occurs over a very narrow temperature range. It is an all-or-nothing type of transition - all the molecules are packed in the "gel" state, or they are all disordered in the liquid crystal phase.
Biological membranes also have this phase transition, but due to their complexity and heterogeneity (lateral phase separation), the transition occurs over a much broader temperature range.
Since the bilayer gets thinner and longer, there is a volume increase in vesicles with the transition to the liquid crystal phase.
Phase transitions can be affected by molecules or ions that interact with lipid bilayers (proteins, Ca2+, detergents, etc.)
Structure of Membrane Proteins
Lipid bilayers comprise the structural barrier that contains cytoplasm. However, it is through the action of membrane proteins that the functionalities such as transport and receptor signalling occur. We will focus here on the integral membrane proteins and a new class of proteins termed the lipid-anchored proteins.
Integral Membrane Proteins
Generally-speaking, these are of two types.
- The first type is essentially a soluble globular protein that has a relatively short hydrophobic amino acid sequence (typically at either the amino or carboxy terminus) that is inserted into the lipid bilayer and essentially keeps the soluble protein anchored to the lipid bilayer.
- The second type of integral protein is almost entirely embedded in the lipid region of the bilayer, with only a small polar portion exposed to the aqueous face of the membrane. This type contains almost exclusively a-helix or b-sheet structures because these can satisfy main chain hydrogen bonding requirements without the need for water molecules (which are not present in the lipid bilayer environment.
Proteins with a hydrophobic anchor
The anchor is often a single a-helix.
- Each amino acid in an a-helix extends the helix by about 1.5Å. Thus, ~20-30 amino acids in an a-helix have a length of approximately 30-45Å and will span most or all of a bilayer
- Glycophorin is an integral membrane protein with an a-helix hydrophobic anchor of 19 amino acids. This is actually observed to span the entire bilayer. Thus, the helix must be somewhat stretched or the bilayer somewhat thinner than 5nm
- Some molecules involved in immune system recognition (i.e. letting the immune system know that the cell is "self") fall into this class of integral membrane proteins (e.g. major transplantation antigen H2).
- Some viral proteins that confer the ability of the virus to invade the cell will have a transmembrane a-helix.
Proteins that are largely embedded
Embedded integral membrane proteins are often globular, as opposed to simply having a linear hydrophobic portion for an anchor. Their globular structure confers their functionality, its just that they require a hydrophobic environment for "solubility".
- They often consist of "up-down" repeats of a-helices, each with a length that essentially spans the bilayer. One example is bacteriorhodopsin. An integral membrane protein in bacteria that is involved in pumping protons out of the cell as part of a light-coupled method of generating energy for the cell. Note: each residue in a helix extends the helix by 1.5Å. With 3.6 residues per turn, each turn of an a-helix is approximately 5.4Å long (0.54nm). Therefore, count the turns in a helix and multiply by 0.54 to get length in nm. A 50nm wide membrane will be spanned by a helix that has about 9 turns or so.
b-sheet secondary structure can also satisfy main chain hydrogen bonding requirement. Therefore, it is also useful in integral membrane protein structures. An example are the porin proteins, which in E. coli are involved in the transport of sugars into the bacteria. Polar and non-polar residues alternate in the sequence forming the b-strands, but the polar residues point inside the structure and the non-polar residues point outside towards the hydrocarbon environment.
Click on the above image to view a VRML file of bacteriorhodopsin
Click on the above image to view a VRML file of a porin molecule
Lipid-Anchored Membrane Proteins
These are soluble globular proteins that have a lipid prosthetic group covalently attached that inserts into the lipid bilayer and anchors the protein to the bilayer
- In some cases the lipid prosthetic group can be reversibly attached, and this can provide a regulatory mechanism if the protein function's requires the participation of integral membrane proteins
Four types of lipid-anchoring motifs have been identified
Amide-linked myristoyl anchors. Myristic acid (14 carbon chain) may be linked via an amide bond to the amino group of the N-terminal glycine residue (known as N-myristoylation) of certain proteins
- Thioester-linked fatty acid acyl anchors. Myristate (14 carbons), palmate (16 carbons), stearate (18 carbons) and oleate (18 carbons, unsaturated) can be thioester linked to cysteine residues in proteins. G-protein coupled receptors are anchored to the lipid bilayer in this way.
- Thioether-linked prenyl anchors. Farnesyl and geranylgeranyl groups are polymers of isoprene units that can be attached to proteins at a cysteine residue. The cysteine to be modified is part of a carboxyl terminal recognition sequence of Cys-Ala-Ala-X. After attachment, a specific protease removes the AAX sequence to leave the carboxyl terminal cysteine with the polyprenyl ether linkage.
- Glycosyl phosphatidylinositol anchors (GPI anchors). These are more complicated structures. They modify the carboxyl terminal amino acid of a protein with an ethanolamine group linked to an oligosaccharide. The oligosaccharide is linked to the inositol group of a phosphatidylinositol. The oligosaccharide comprises a tetrasaccharide core (3 mannose, 1 glucosamine). Various derivatives of this basic organization are found. GPI anchors are unique to animals.
© 2001 Dr. Michael Blaber