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Lecture 07 (Chapter 12) Membrane Proteins. Proteins that interact with biological membranes. Targets of over 50% of all modern medicinal drugs. Approximately.

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Presentation on theme: "Lecture 07 (Chapter 12) Membrane Proteins. Proteins that interact with biological membranes. Targets of over 50% of all modern medicinal drugs. Approximately."— Presentation transcript:

1 Lecture 07 (Chapter 12) Membrane Proteins

2 Proteins that interact with biological membranes. Targets of over 50% of all modern medicinal drugs. Approximately 20–30% of all genes in most genomes encode membrane proteins. Overington JP, Al-Lazikani B, Hopkins AL (December 2006); Krogh, A.; Larsson, B. R.; Von Heijne, G.; Sonnhammer, E. L. L. (2001);, A.Von Heijne, G.

3 Membrane Protein Functions Serve as mediators between cell and the extracellular region, or interior of an organelle and the cytosol. Transport metabolites and ions across the cell membrane. Convert energy from light into chemical and electrical energy, and couple flow of electrons to ATP synthesis. Act as signal receptors and transduce signals across membrane (e.g., neurotransmitters, growth factors, hormones, light or chemotactic stimuli). Perform various enzyme functions. Cell recognition and communication.

4 Membrane Protein Classifications Integral membrane proteins – These are permanently attached to membrane Lipid-linked proteins – These are covalently bonded to a fatty acid such as palmitate or myristate and serves to anchor the protein to the cell membrane. Peripheral membrane proteins – These are temporarily attached to membrane or to integral membrane proteins

5 Fluid Mosaic Model of Lipid Bilayer

6 Crystallization of Membrane Proteins Difficult to crystallize because: – Insoluble in aq. Buffers (Due to hydrophobic surface regions) – Tend to denature in organic solvents Can be solubilized using aqueous detergents, then purified in native state. How this works: Amphipathic detergent adheres to hydrophobic surface of protein, so protein- detergent complex becomes hydrophilic. Can be crystallized in this state, but frequently produces low resolution crystals

7 2D Crystals of Membrane Proteins 2D crystals of membrane proteins can be studied using electron microscopy. Bacteriorhodopsin is a 248 residue, integral membrane protein that binds retinal and uses light energy to transport protons across the membrane. Bacteriorhodopsin is a 248 residue, integral membrane protein that binds retinal and uses light energy to transport protons across the membrane Bacteriorhodopsin was first visualized using this method in 1976 (7 Å resolution) – Observed to have 7 transmembrane α-helices. – Helices confirmed in 1990 at 3 Å resolution. – Structure now available at 2 Å resolution. Structural studies of bacteriorhodopsin provided information used to predict transmembrane helices from primary sequence.

8 Integral Membrane Proteins Functions – Transfer information – Transport material – Energy conversion Types – Type I: Single transmembrane span, N-terminus in ectodomain, C- terminus in cytosol – Type II: Single span, C-terminus in the ectodomain, N-terminus in the cytosol – Type III: Multiple spans – Type IV: Several different polypeptides assembled to form a channel – Type V: Lipid-linked protein – Type VI: Proteins with both transdomain component and lipid anchor Nelson, D. L., & Cox, M. M. (2008). Principles of Biochemistry (5th ed., p. 377).

9 Integral membrane proteins - glycoproteins Proteins that contain oligosaccharide chains (glycans) covalently attached to AA side chains as co- or post- translational (N- or O-) glycosylation. Function as receptors.

10 Integral membrane proteins - sialoglycoproteins Glycoproteins combined with sialic acid. Glycophorins (A –D) are sialoglycoproteins found in the membranes of red blood cells. 1afo Glycophorins A and B: Bear antigenic determinants for MN and Ss blood groups. Glycophorins C and D: Maintains erythrocyte shape and regulates membrane properties. Acts as receptor for Plasmodium falciparum protein.

11 Porins/Ion Channels Porins 16 Strand, antiparallel up and down β-barrel proteins that form trimer to traverse the cell membrane. Sequence composed of alternating hydrophobic and hydrophilic residues: hydrophobic residues face the lipid membrane, hydrophilic residues form aqueous channels for molecules to pass through the membrane. ucrose_specific_porin_1A0S.png The porin channel Channel blocked by loop (the eyelet) which limits size of solute that can pass Channel is lined with charged amino acids (positive on 1 side, negative on the other) – Ion selectivity. Calcium ions bind in channel to balance excess negative charges Found in outer membrane of Gram- negative bacteria, mitochondria and the chloroplast

12 Ion/Potassium Channels Allow potassium, sodium, calcium and chloride ions to diffuse across lipid bilayer to balance differences in electrical charge (membrane potential) between the 2 environments. The membrane potential of resting cells is regulated primarily by potassium ions moving through potassium channels. Zhou Y, Morais-Cabral JH, Kaufman A, MacKinnon R (2001)

13 Potassium Channels - Overview Most widely distributed type of ion channel Found in virtually all living organisms and most types of cells Span cell membranes to allow passage of K+ into cells Potassium channels conduct potassium ions down their electrochemical gradient, doing so both rapidly (up to the diffusion rate of K+ ions in bulk water) and selectively (by a factor of 10, 000 over Na+, despite the sub-angstrom difference in ionic radii). Potassium channels conduct potassium ions down their electrochemical gradient, doing so both rapidly (up to the diffusion rate of K+ ions in bulk water) and selectively (by a factor of 10, 000 over Na+, despite the sub-angstrom difference in ionic radii). Biologically, these channels act to set or reset the resting potential in many cells. In excitable cells, such as neurons, the delayed counterflow of potassium ions shapes the action potential. By contributing to the regulation of the action potential duration in cardiac muscle, malfunction of potassium channels may cause life-threatening arrhythmias. Potassium channels may also be involved in maintaining vascular tone. They also regulate cellular processes such as the secretion of hormones (e.g., insulin release from beta-cells in the pancreas) so their malfunction can lead to diseases (such as diabetes).

14 Potassium Channels - Types Calcium-activated potassium channels - Open in response to the presence of calcium ions or other signalling molecules. Inwardly rectifying potassium channels – Pass positively-charged current more easily into the cell. Tandem pore domain potassium channel - "leak channels" regulated by oxygen tension, pH, mechanical stretch, and G-proteins. These set the negative membrane potential of neurons. Voltage-gated potassium channels - Open or close in response to changes in the transmembrane voltage.

15 Calcium-activated potassium channels Based on conductance (big, intermediate, and small), can be sub-divided into: BK channels IK channels SK channels

16 Calcium-activated BK potassium channels BK channels (Big Potassium), also called slo1, are characterized by large conductance of potassium ions (K + ) through cell membranes. Channels are activated by changes in membrane electrical potential and/or by increases in intracellular [Ca 2+ ]. Essential for the regulation of several physiological processes including smooth muscle tone and neuronal excitability. An overview of the potassium channel familyAn overview of the potassium channel family, Miller, 2000; Calcium-Activated Potassium Channels and the Regulation of Vascular Tone, Ledoux et al., 2006 A: structure of the α and β1-subunits of the BK channel. The β1-subunit consists of 2 transmembrane domains and the α-subunit of 11 (S0–S10) hydrophobic domains, with S0–S6 located in the cytoplasmic membrane and the pore region (P) between S5 and S6. B: Association of four α and four β1-subunits forms the native BK channel.

17 Calcium-activated IK potassium channel The IK channel (KCa3.1) is expressed mainly in peripheral tissues such as those of the haematopoietic system, colon, placenta, lung and pancreas. Has been implicated in a wide range of cell functions, including vasodilation of the microvasculature, K + flux across endothelial cells of brain capillaries and phagocytic activity of neutrophils. In comparison with the large-conductance (BK) channels, KCa3.1 is much more sensitive to Ca 2+ than BK channels. – This high affinity is due to the presence of 4 calmodulin molecules bound to the cytoplasmic tails of the four pore-forming α-subunits. – Before the channel can open, Ca 2+ must bind to each of the calmodulins to induce the co-operative conformational change that opens the gate. Cross-section of the IK channel. Binding of calcium ion to CaM produces conformational change that opens the pore.

18 Calcium-activated SK potassium channels Small single channel conductance. SK channels allow potassium ions to cross the cell membrane and are activated (opened) by an increase in the intracellular [Ca 2+ ] through N-type calcium channels. SK channels are thought to be involved in synaptic plasticity and therefore play important roles in learning and memory. Calcium-Activated Potassium Channels and the Regulation of Vascular Tone, Ledoux et al., 2006; institutes/vollum/faculty/adelmanlab.cfm SK channels are Ca-activated K channels, gated solely by intracellular Ca ions. SK channels are constitutively associated with calmodulin (CaM) that binds to the CaMBD in the intracellular C- terminus of the channels. When Ca ions bind to the N-lobe E-F hands of CaM, a gating transition is initiated, the gate of the channel opens and K flows through the channel pore, exerting a repolarizing influence on the membrane potential.

19 Inwardly rectifying potassium channels The voltage-dependent block by polyamines (i.e., spermine, spermadine) and magnesium ions from within the cell directs positive current (K+) inwards. This current may be involved in regulation of neuronal activity, by stabilizing resting membrane potential of the cell. This mechanism remains to be determined. 1p7b Structural insights into gating and the formation of a macromolecular GIRK signalling complex, Nature Reviews, 2010; There are seven subfamilies of Kir channels, denoted as Kir1 - Kir7. Each subfamily has multiple members that have nearly identical AA sequences across known mammalian species. Kir channels are formed as homotetrameric membrane proteins. Each of the 4 identical protein subunits consists of 2 membrane- spanning alpha helices (M1 and M2).

20 Tandem pore domain potassium channel Tandem pore domain potassium channels are a family of 15 members form what is known as "leak channels“. Leak channels allow potassium to exit the cell in order to reduce positive charge within the cell and maintain stable resting membrane potential. Channels are regulated by several mechanisms including: – oxygen tension – pH – mechanical stretch – G-proteins For most, the α subunits consist of 4 transmembrane segments, each containing 2 pore loops. They structurally correspond to two inward-rectifier α subunits and thus form dimers in the membrane. K2P dimer

21 Voltage-gated potassium (Kv) channels - Overview Present in all animal cells. Open and close based on voltage changes in the cell's membrane potential. During action potentials, these channels open and allow passive flow of K + ions from the cell to return the depolarized cell to a resting state. Kv channels are one of the key components in generation and propagation of electrical impulses in nervous system.; Voltage-gated potassium channels as therapeutic targets, Wulff et al., 2009

22 Voltage-gated potassium (Kv) channels - Overview The tetrameric structure of Kv channels is made of two functionally and structurally independent domains: an ion conduction pore, and voltage-sensor domains. The ion conduction pore is made of four subunits which are arranged symmetrically around the conduction pathway. Voltage-sensor domains are positioned at the periphery of the channel and consist of four transmembrane segments (S1-S4). Structural rearrangement of the voltage-sensor domains in response to changes in the membrane potential, and in particular S4, which includes positively charged amino acids at every third position, results in conformational changes in the conduction pore, which could open or close the ion conduction pathway. The nature of these movements and conformational changes in the voltage-sensors have been subject to controversy and several models for voltage-gating have been proposed. Ion channels: A paddle in oil, Lee, 2006;

23 Calcium channels - Overview Two types of ion channels with selective permeability to calcium ions I.Voltage gated calcium channels I.L-type (High voltage activated) I.Found in muscle, bone, ventricular myocytes II.P-type (High voltage activated) I.Found in Purkinje neurons in cerebellum III.N-type (Hight voltage activated) I.Found in brain and peripheral nervous system IV.R-type (Intermediate voltage activated) I.Found in cerebellar granule cells, other neurons V.T-type (Low voltage activated) I.Found in neural cells, bone II.Ligand gated calcium channels I.IP3 receptor II.Ryanodine receptor III.Two pore channels IV.Cation channels of sperm V.Store operated channels Ion channels: A paddle in oil, Lee, 2006;

24 L-type high-voltage-activated Calcium channels Voltage-gated calcium channels in the human adrenal and primary aldosteronism. J Steroid Biochem Mol Biol., Felizola et al., 2014; Calcium channels — basic aspects of their structure, function and gene encoding; anesthetic action on the channels — a review. Can J Anaesth., Yamakage M, Namiki A, 2002 L-type calcium channels remain activated for longer periods of time. Channel is comprised of four protein subunits (Cav1.1, Cav1.2, Cav1.3, Cav1.4). Responsible for excitation-contraction coupling of skeletal, smooth, cardiac muscle and for aldosterone secretion in endocrine cells of the adrenal cortex. In cardiac myocytes, the L-type calcium channel direct Ca2+ current into the cell and triggers calcium release from the sarcoplasmic reticulum (SR) by activating Ryanodine receptor 2 (RyR2).

25 L-type high-voltage-activated Calcium channels The alpha-1 subunit forms the pore for the import of extracellular calcium ions and, though regulated by the other subunits, is primarily responsible for the pharmacological properties of the channel [PMID: 11031246]. It shares sequence characteristics with all voltage-dependent cation channels, and exploits the same 6- helix bundle structural motif - in both sodium and calcium channels, this motif is repeated 4 times within the sequence to give a 24-helix bundle. Within each of these repeats, 5 of the transmembrane (TM) segments (S1, S2, S3, S5, S6) are hydrophobic, while the other (S4) is positively charged and serves as the voltage-sensor. Calcium channels — basic aspects of their structure, function and gene encoding; anesthetic action on the channels — a review. Can J Anaesth., Yamakage M, Namiki A, 2002; Voltage-Gated Calcium Channel Antagonists and Traumatic Brain Injury, Gurkoff et al., 2013

26 IP3 Receptor Inositol trisphosphate (IP3) receptor is a membrane glycoprotein complex acting as a Ca 2+ channel activated by inositol trisphosphate (InsP3). IP3R is necessary for the control of cellular and physiological processes including: – cell division – cell proliferation – apoptosis – fertilization – development – behavior – learning – memory IP3R represents a dominant second messenger leading to the release of Ca 2+ into the cytosol from intracellular store sites (i.e., SR lumen). Structure of the inositol 1,4,5-trisphosphate receptor binding core in complex with its ligand, Bosanac I, Alattia JR, Mal TK, et al., Nature, 2002; IP3 binding core (IBC) of IP3 receptor with bound IP3 molecule Acting on a membrane phospholipid, phospholipase C cleaves off IP3, which is a small polar molecule. IP3 binds to IP3R which opens to release calcium into the cytosol.

27 Ryanodine Receptor 1 RYR1 functions as an SR calcium release channel, as well as a connection between the sarcoplasmic reticulum and the transverse tubule. Found primarily in skeletal muscle. Triggers muscle contraction following depolarization of T-tubules. Repeated very high-level exercise increases the open probability of the channel and leads to Ca 2+ leaking into the cytoplasm. Can also mediate the release of Ca 2+ from intracellular stores in neurons, and may thereby promote prolonged Ca 2+ signaling in the brain. Required for normal embryonic development of muscle fibers and skeletal muscle. Required for normal heart morphogenesis, skin development and ossification during embryogenesis.

28 Lipid-linked proteins: G proteins G proteins, (guanine nucleotide-binding proteins), are a family of proteins that act as molecular switches inside cells. Transmit signals from a variety of stimuli outside a cell to the inside. Their activity is regulated by factors that control their ability to bind to and hydrolyze guanosine triphosphate (GTP) to guanosine diphosphate (GDP). When they bind GTP, they are 'on', and, when they bind GDP, they are 'off'. G proteins belong to the larger group of enzymes called GTPases. (1) ligand activates the G protein-coupled receptor. (2) Induces a conformational change in the receptor that allows the receptor to function as a guanine nucleotide exchange factor (GEF) that exchanges GTP for GDP (4) - thus turning the GPCR "on". The GTP (or GDP) is bound to the Gα subunit in the traditional view of heterotrimeric GPCR activation. (5)This exchange triggers the dissociation of the Gα subunit (which is bound to GTP) from the Gβγ dimer and the receptor as a whole. Gα subunit stimulates production of cAMP from ATP.

29 Peripheral membrane proteins Adhere only temporarily to the biological membrane with which they are associated (reversible binding). Attach to integral membrane proteins, or penetrate the peripheral regions of the lipid bilayer. – May be regulatory protein subunits of many ion channels and transmembrane receptors. Tend to collect in the water-soluble component, or fraction, of all the proteins extracted during a protein purification procedure. – Proteins with GPI anchors are an exception to this rule and can have purification properties similar to those of integral membrane proteins. Reversible binding of these proteins is associated with regulation of cell signaling and other cellular events. These proteins may interact with membrane in several ways: 1.By amphipathic α-helix parallel to membraneα-helix 2.By a hydrophobic loop 3.By a covalently bound membrane lipid (lipidation) 4.By electrostatic or ionic interactions with membrane lipids (e.g. through a calcium ion)ionic interactions

30 Categories of peripheral membrane proteins Enzymes – Phospholipase C: Hydrolyzes PIP2 into IP3 and diacylglycerol Membrane-targeting domains (lipid clamps) – C2 domains: Bind phosphatidylserine or phosphatidylcholine Structural domains – Annexins: Calcium-dependent intracellular membrane/phospholipid binding. Involved in membrane fusion and ion channel formation. Transporters of small hydrophobic molecules – Glycolipid transfer protein Electron Carriers – Cytochrome C: Transfers electrons between Complex III (CoQ-Cyt C reductase) and Complex IV (Cyt C oxidase) in the electron transport chain. Polypeptide hormones, toxins and antimicrobial peptides – Venom toxins (e.g., snake venom)

31 Phospholipase C Enzymes that cleave phospholipids just before the phosphate group. PLC cleaves the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) into diacyl glycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). DAG remains bound to the membrane. IP3 is released as a soluble structure into the cytosol, where it binds to IP3 receptors in the smooth endoplasmic reticulum (ER), releasing calcium into cytosol, causing a cascade of intracellular changes. Calcium and DAG also work together to activate protein kinase C, which goes on to phosphorylate other molecules, leading to altered cellular activity. "PLC role in IP3-DAG pathway" by RaihaT - Own work. Licensed under CC BY-SA 3.0 via Wikimedia Commons -

32 C2 Domains Protein structural domains that help target proteins to cell membranes. Typical structures consist of a beta-sandwich with 8 strands that may bind 2-3 calcium ions. C2 domains frequently bind with enzyme domains. – Example: The C2 domain in PTEN, brings the phosphatase domain into contact with the plasma membrane, where it can dephosphorylate its substrate, phosphatidylinositol (3,4,5)-trisphosphate (PIP3), without removing it from the membrane.; Synaptotagmin binds phospholipids in a calcium dependent manner. Synaptotagmin's C2 domain shows a single calcium-binding site made up of residues from two loops at one end of the domain (red spheres). PI-PLC delta 1 also has calcium binding between the analogous loops (cyan spheres). However, PI-PLC has two prominent calcium binding sites in this location. The presence of these sites and the orientation of this domain with respect to the catalytic domain suggest that the C2 domain may be important for interacting with phospholipid head groups of the membrane.

33 Annexins Over 160 different types in 65 species. Bind negatively charged phospholipids in a calcium dependent manner. Composed of two major domains. – Core region at the COOH terminal consisting of alpha helical disc with type 2 calcium binding sites. These interact with membrane phospholipids. – Core domain (310 AAs) made up of 4 similar repeats (annexin repeats) 5 alpha helices, 70 AAs long. – Head region at NH2 terminal located on the concave side of the core region which provides a binding site for cytoplasmic proteins. This may become phosphorylated in some annexins and can cause affinity changes for calcium in the core region or alter cytoplasmic protein interactions. Annexins: form structure to function, Gerke, V. & Moss, S, Physiol. Rev. 2002 1axn: Human Annexin III Annexin participate in: Vesicle transport Membrane organization Mitosis Inflammation reduction Apoptosis

34 Glycolipid Transfer Protein Cytosolic protein that accelerates the intermembrane transfer of various glycolipids. Catalyzes the transfer of various glycosphingolipids between membranes but does not catalyze the transfer of phospholipids. May be involved in the intracellular translocation of glucosylceramides. Has been found in brain, kidney, spleen, lung, cerebellum, liver and heart. 1swx Glycosphingolipid binding specificity is achieved through recognition and anchoring of the sugar-amide headgroup to the GLTP recognition center by hydrogen bond networks and hydrophobic contacts, and encapsulation of both lipid chains, in a precisely oriented manner within a 'molded-to-fit' hydrophobic tunnel.

35 Cytochrome C Cytochrome c is a component of the electron transport chain in mitochondria. The heme group of cytochrome c accepts electrons from the bc1 complex and transfers electrons to the complex IV. Cytochrome c is also involved in initiation of apoptosis. Upon release of cytochrome c to the cytoplasm, the protein binds apoptotic protease activating factor-1 (Apaf-1). Can catalyze several reactions such as hydroxylation and aromatic oxidation, and shows peroxidase activity by oxidation of various electron donors. Entrez Gene: Cytochrome C;


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