PHA111 Membrane Bound Proteins.pdf

MPharm Programme Membrane Bound Proteins Dr. Mark Gray Slide 1 of 24 MPharm PHA111 Membrane Bound Proteins Signalling Pathways Slide 2 of 24 MPharm PHA111 Membrane Bound Proteins Reversible phosphorylation is key to biological signalling R H N O N H OH R' Kinase R H N O N H O O P...

MPharm Programme Membrane Bound Proteins Dr. Mark Gray Slide 1 of 24 MPharm PHA111 Membrane Bound Proteins Signalling Pathways Slide 2 of 24 MPharm PHA111 Membrane Bound Proteins Reversible phosphorylation is key to biological signalling R H N O N H OH R' Kinase R H N O N H O O P O • Kinases (phosphotransferases) catalyse the reversible phosphorylation of specific serine, threonine and tyrosine O and histidine residues within other proteins. • The resulting change in sterics and electronics affects protein tertiary and quaternary structure, as well as protein-protein interactions. This can lead to modulation of enzymatic activity. • Kinases are a key target for new anti-cancer drugs. Slide 3 of 24 MPharm PHA111 Membrane Bound Proteins R' The fluid mosaic model Slide 4 of 24 MPharm PHA111 Membrane Bound Proteins Integral membrane proteins • Types I and II differ in domain orientation. • Multiple transmembrane helicies within a single polypeptide for type III, whereas type IV involves multiple polypeptide chains. • Types V and VI have covalent lipid anchors. • Most transmembrane helicies have around 6-7 turns. Slide 5 of 24 MPharm PHA111 Membrane Bound Proteins Lipid anchors for membrane proteins Slide 6 of 24 MPharm PHA111 Membrane Bound Proteins Glycophorin • Erythrocyte glycoprotein, each hexagon represents a tetrasaccharide. • Function not known, loss of sugars results in destruction of red blood cell. • Main transmembrane domain (Leu75-Tyr93) consists of 19 amino acids in an α-helix. • Harder to get high quality structural information for membrane bound proteins than freely soluble ones. Slide 7 of 24 MPharm PHA111 Membrane Bound Proteins Bacteriorhodopsin, PDB ID 2AT9 Slide 8 of 24 MPharm PHA111 Membrane Bound Proteins Slide 9 of 24 MPharm PHA111 Membrane Bound Proteins Slide 10 of 24 MPharm PHA111 Membrane Bound Proteins Hydropathy Plots • The average hydropathy of 7-amino acid sequences is taken and plotted. • The hydropathy of the window at residue 4 is the average of the individual hydropathies of residues 17. • Similarly the hydropathy of the residue 5 window looks at residues 2-8. • Positive hydropathy windows for 20 residues in a row are indicative of a transmembrane domain. Slide 11 of 24 MPharm PHA111 Membrane Bound Proteins A closer look at amino acids at the water-lipid interface 1BL8 1AF6 1QD5 Streptomyces lividans 1QD5 1PHO E. coli • Tryptophan (red, hydropathy -0.9) and tyrosine (orange, hydropathy -1.3) are often found at the water-lipid interface. • Charged residues (blue) are usually found exposed to water. Slide 12 of 24 MPharm PHA111 Membrane Bound Proteins GLUT1: a glucose transporter protein Slide 13 of 24 MPharm PHA111 Membrane Bound Proteins Helix bundling allows for hydrogen bonding to glucose in channel region. Slide 14 of 24 MPharm PHA111 Membrane Bound Proteins E. coli lactose permease • a) X-ray structure, PDB ID 1PV7. b) hypothetical second conformation for lactose transport. • Switching is believed to be due to change in protonation in the Glu325-Arg302 salt bridge according to transmembrane proton gradient. Slide 15 of 24 MPharm PHA111 Membrane Bound Proteins The calcium pump of sarcoplasmic reticulum. PDB ID 1EUL • Single polypeptide with Mr ~100,000. • Phosphorylation of Asp351 is believed to cause a widespread conformational change, changing the exposure face of the calcium binding domain. • This conformational change also affects the affinity for calcium in its binding site, allowing release into the lumenal side of the membrane. Slide 16 of 24 MPharm PHA111 Membrane Bound Proteins ATP-binding cassette transporters (ABC Transporters) pump ions, biomolecules and drugs in and out of cells lipid A flippase 1JSQ E.coli vitamin B12 importer 1L7V • Human ABC transporter MDR1 responsible for tumor chemotherapy resistance to drugs such as adriamycin, doxorubicin and vinblastine. • Microbial ABC transporters are targets in design of new antibiotics. Slide 17 of 24 MPharm PHA111 Membrane Bound Proteins Cystic Fibrosis Slide 18 of 24 MPharm PHA111 Membrane Bound Proteins AQP-1, an aquaporin. PDB ID: 1J4N • Tetramer of identical subunits, each subunit has one water permeable pore. • Key amino acid sequence Asn-Pro-Ala (NPA), conserved in all aquaporins. • c) Hydrophilic atoms (mainly backbone carbonyls) red, simulated water molecules orange, Phe58 blue. • Size based specificity filter, consisting of Phe58, His182, Cys191, & Arg197. Arg & His hydrogen bond to water but repel hydronium. Slide 19 of 24 MPharm PHA111 Membrane Bound Proteins A potassium channel from Streptomyces lividans PDB ID 1BL8 • Microbial cells require maintenance of a high concentration gradient of K in order to survive. • Eight transmembrane α-helicies, two each from four subunits. Cone shape. Discriminates for K based on size and shape. Slide 20 of 24 MPharm PHA111 Membrane Bound Proteins A closer look at potassium transport PDB ID 1J95 Slide 21 of 24 MPharm PHA111 Membrane Bound Proteins β-barrel membrane proteins 1FEP • • 1QD5 1MAL Usually 20 or more lines of β-sheet coming together to maximise secondary structure interactions. More extended conformation means that a sequence of only 7hydrophobic residues is enough to span a membrane. Slide 22 of 24 MPharm PHA111 Membrane Bound Proteins 1EK9 7AHL Valinomycin: a potassium binding antibiotic • Valinomycin selectively binds K via its carbonyl oxygen atoms, The valine-like side chains allow the complex to pass readily through the lipid bilayer. • This allows the concentration of K either side of the membrane to equilibrate, killing the cell. Slide 23 of 24 MPharm PHA111 Membrane Bound Proteins Summary • Membrane bound proteins usually serve as receptors, but may also display enzymatic activity. • These proteins pass along messages through physical changes, as in the reversible phosphorylation of kinases or through conformational changes. • These changes in shape determine which partner substances are allowed to bind, thus passing on the ‘message’. • In some cases these changes can allow for the controlled passage of substances into or out of the cell. • Certain antibiotics act by disrupting the fine control of these processes. 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