Lecture 15. Membrane Proteins I

Transcription

1 Lecture 15 Membrane Proteins I Introduction What are membrane proteins and where do they exist? Proteins consist of three main classes which are classified as globular, fibrous and membrane proteins. A cell is enveloped by a membrane which makes the boundary of a cell and enables it to maintain the distinction between cytosolic and extracellular milieu. Cells consist of various organelles such as golgi body, endoplasmic reticulum, mitochondria and several other membrane bound organelles. The difference between cytosol and these organelles are maintained by individual membranes. These biological membranes are made up of mainly lipid bilayers whereas functions are carried out by membrane proteins. Classification of membrane proteins: Membrane associated proteins can be classified in the following two ways: 1. Mode of interaction with the membranes 2. Cellular locations According to the literature, the membrane proteins can be categorized as follows: 1. Type I membrane proteins 2. Type II membrane proteins 3. Multipass transmembrane proteins 4. Lipid chain anchored-membrane proteins 5. GPI-anchored-membrane proteins 6. Peripheral membrane proteins 1

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3 Integral or intrinsic membrane proteins Integral membrane proteins are associated with membranes and interact strongly with the hydrophobic part of the phospholipid bilayer. Presence of one or more apolar regions accounts for the span of lipid bilayer (α-helix and β-sheet as well). They interact mainly through van der Waals interaction with the hydrophobic core of the lipid bilayer. Thus they can be extracted from the membrane only through membrane disruption by detergents. Examples: GPCRs, rhodposins proteins etc. Peripheral or extrinsic membrane proteins Peripheral or extrinsic membrane proteins are known to interact either non covalently with the membrane surface through electrostatic or hydrogen bonds or with covalent bonds through lipids or GPI (glycosylphosphatidylinositol) anchors [Fig. 2 (d)]. They interact with the hydrophilic surfaces of the bilayer through electrostatic interaction. They can be isolated from the membrane using strong salt or change in ph. Examples: Cytochrome C protein. Type I membrane proteins This is a single-pass transmembrane protein. The N-terminus of this protein is extracellular (luminal) and C-terminus remains in the cytoplasmic region for a cell (or organelle) membrane. [Fig. 1 (a)] Type II membrane proteins This is a single-pass transmembrane protein. The C-terminus of this protein is extracellular (luminal) and N-terminus remains in the cytoplasmic region for a cell (or organelle) membrane. [Fig. 1 (b)] Multipass transmembrane proteins Multipass transmembrane proteins [Fig. 2 (a)] are able to cross the lipid bilayer multiple times compared to Type I and Type II single pass membrane proteins [Fig. 1 (a) and (b)] which can cross the lipid bilayer only once. Membrane straddling region of polypeptide chains possess mostly α-helical conformation as in the lipid environment hydrogen bonding between polypeptide chains would be maximum if it form helical conformation. Lipid chain anchored-membrane proteins Lipid chain anchored-membrane proteins [Fig. 2 (b)] are related with lipid bilayer via one or greater than one covalently attached fatty acid chains or prenyl groups (other type of lipid chains). GPI-anchored-membrane proteins GPI-anchored-membrane proteins [Fig. 2 (c)] are associated with lipid bilayer via glycosylphosphatidylinositol (GPI) anchor. 3

4 Structure of membrane proteins Biological membranes Before going into the details of the membrane proteins we need to look at the structural aspect of the biological membranes. Biological membranes were considered to be two dimensional fluids consist of two leaflets which comprised of mainly lipid molecules. According to the fluid mosaic model, the outer part is made up of hydrophilic (ionic and polar head groups) groups which interact with the aqueous solvents. The inner part is comprised of hydrocarbon chains of the lipid. The fluid mosaic model considers membrane as a dynamic system where both proteins and lipids can move and interact. Role of length and magnitude of dielectric gradient of membrane bilayer thickness The variation in the dielectric constant between the ionic part and the hydrophobic part (~80 to 2 Debye) is significant which occurs over a comparatively short distance and thus cover up of charge or leaving a hydrogen bond unsatisfied is unfavored. Peptide backbone of a protein is comprised of polar amino and carbonyl groups and thus covering of a peptide backbone in the membrane interior leaving the hydrogen bonding unsatisfied is energetically unfavorable. 4

5 Thickness of the bilayer governs the length of low dielectric well which in turn determines interior, exterior and interfacial regions of proteins. Only specific conformations of proteins get stabilized according to the bilayer thickness. There should be comparable length factor between the hydrophobic thickness of the bilayer and the hydrophobic length of membrane protein. It further regulates self aggregation of protein to minimize the unfavorable interactions if the comparable relationship is absent. Hydrophobic chain packing plays an important role in stabilizing protein structure. The favored arrangement of the hydrophobic chains of bilayer is when they are aligned to each other maximizing van der Waals interactions. Thus, cylindrical shape of the protein will be able to minimize number of lipid chains disordered by their presence and also area of the proteins exposed to the bilayer. 5

6 Structural Classifications: Primary, Secondary, Tertiary and Quaternary Primary Structure: The interior of the membrane is nonpolar in nature. Therefore, surface residues of transmembrane proteins are expected to be nonpolar in nature so that they can reside in the interior part of the membrane. A hydrophobicity scale was formulated to assign numerical values to the hydrophobic nature of amino acid side chains for the prediction purpose. Numerous scale such as Kyte-Doolittle scale, GES scale, etc have been introduced to postulate which part of the proteins will reside in the inner part of the membrane. Secondary Structure The inner part of the membrane is devoid of water. Thus the only possibility of the atoms of the peptide backbone to undergo hydrogen bonding is either side chain atoms or other atoms on the peptide backbone. The most favored arrangements are α-helical or β-sheet arrangement as regular arrays of hydrogen bonding occurs between amide nitrogen and carbonyl oxygen atoms. Most of the membrane proteins are found to be helical in nature. This preference for helical structure over β-sheet arrangement might be due to the following reasons: 1. Helix length is sufficient to accommodate any little changes in bilayer thickness 2. Individual insertion of helices is possible whereas before insertion β-strands have to be aligned or zipper up to form sheets Tertiary Structure Folding of integral membrane proteins occurs via a two step process. This picture is clear from the structure of transmembrane helical protein glycophorin. In the first step insertion and formation of helices followed by association of transmembrane helices in the second step. In case β-sheet proteins, first formation of β-sheet occurs followed by insertion in the membrane. Similar type of packing is observed in the packing of membrane proteins, hydrogen bonding between the helices is less in number and salt bridges are absent. 6

7 Mechanism of association of transmembrane helices in the membrane interior Two proposed mechanisms are there: 1. Arrangement of nonpolar side chains of proteins takes place resulting maximum packing of helices 2. Polar and hydrogen bonding side chains of proteins arrange such that they will stabilize interaction between the helices As rise/residue is ~3.6 for a helix, thus atleast one residue should be polar for interaction between two helices and two or more residues for multiple helix interaction. To explore which side of the helix interact with the membrane interior and which side interacts with other helix a term hydrophobic moment has been introduced by Eisenberg and co-workers. The hydrophobic moment is a vector of the sum of the hydrophobicity of the particular residues on the helix times the unit vector from the nucleus of the α-carbon to the center of the side chain. 3. Helical moment which arises due to the particular configuration of peptide bonds in helix structure that favors association of helices. Peptide bonds have weak dipole which arises due to their resonance structure. Alignment of these moments within helix results in an overall moment. Antiparallel structure of helix is preferred over parallel structure. More the length of the helix more will be the moment. It has been observed that transmembrane helices have mostly antiparallel configuration. 4. Optimal packing of the helices also governs association of the helices in the bilayer. Angle between membrane spanning proteins and bilayer plane is ~21 which is 20 for the angle between two membrane spanning proteins. A knob-into-hole packing arrangement of helices results left-handed coiled-coil arrangement of proteins. 7

8 Quaternary structure Membrane protein oligomerization has been explored by measuring the dimerization of the single transmembrane protein glycophorin with the association of bacteriorhodopsin helices in bilayers. The significance of packing interactions between the helices was recognized. Our knowledge about the thermodynamics, kinetics and several other physical properties that direct the oligomerization of integral membrane proteins is inadequate due to the lack of methods to monitor protein-protein interaction in the membrane. 8