Slide 1A protein’s surface polarity corresponds to its environmentTransmembrane regions are usually α-helices or continuous β-sheets (β-barrels)Slide 4In integral transport proteins, interiors are hydrophilic and exteriors are hydrophobicTransporters catalyze passage through the membraneSlide 7Lipid-linked proteins cluster in or outside of rafts based on their linked lipidMethods for determining protein structureSlide 10A protein binds a ligand through a specific, reversible interactionThe association constant (Ka) and disassociation constant (Kd)provide a measure of affinity between protein & ligandThe fraction of occupied binding sites (θ) is proportional to the ligand concentrationA protein with higher affinity for a ligand has a higher binding curve and lower KdSlide 15The sequential (gradual) model of cooperativity Subunits can adopt multiple conformations; Binding of ligand (S) induces conformational changes in the bound subunit and in neighboring subunits; Bound conformations may have higher or lower affinity for ligand than the free protein.The oxygen-binding curves of Mb and Hb reflect their different functionsHeme cofactors bind O2The proximal His links flattening of the heme to shifting of helix F in the T R transition.Modulators/Effectors of O2 bindingSlide 21EnzymesSlide 23Enzymes use several catalytic mechanisms (often together) to enhance reaction ratesSlide 25Slide 26Slide 27Slide 28Slide 29Slide 30Slide 31Slide 32Slide 33Slide 34Slide 35Slide 36Slide 37Slide 38Slide 39Slide 40Slide 41Slide 42Slide 43Slide 44Slide 45Slide 46Membrane Proteins:1. Integral proteins: proteins that insert into/span the membrane bilayer; or covalently linked to membrane lipids. (Interact with the hydrophobic part of the membrane)2. Peripheral proteins: interact with integral protein through non-covalent interaction; or interact with polar head groups of the membrane lipids. (charge interaction is common)3. Amphitropic proteins: Associate with membrane conditionally. Usually subjected to biological regulation.A protein’s surface polarity corresponds to its environmentAlso, often ‘positive inside’ – positively charged aa’s facing cytoplasmic regionTyr and Trp exhibit ‘snorkeling’ – pointing their polar group toward mb exteriorTransmembrane regions are usually α-helices or continuous β-sheets (β-barrels)Bacteriorhodopsin: a light-driven proton pumpPorin: a pore-forming proteinBackbone hydrogen bonds can be self-satisfied.transmembranehelixTransmembrane helices are predicted by hydrophobic stretches of 20-25 aa residuesIn integral transport proteins, interiors are hydrophilic and exteriors are hydrophobicGlucose transporterTransporters catalyze passage through the membraneLipid-linked proteins cluster in or outside of rafts based on their linked lipidMethods for determining protein structure•Sequence:–Edman degradation: Remove one modified a.a from N-terminus at a time;–Mass spectrometry: Generate small fragments and measure the M/Z ratio. •Secondary structure:–Circular Dichroism–FTIR•Tertiary, quaternary structure:–NMR: derived distance constraints are used to calculate likely protein conformations–X-ray crystallography: Electron density map allows for positioning of protein atoms, revealing structureIndicate the composition of secondary structuresOH-N++--A protein binds a ligand through a specific, reversible interactionProteinbindingsiteProtein-ligandcomplexPotential ligands:(any atom or molecule, including a protein)The association constant (Ka) and disassociation constant (Kd)provide a measure of affinity between protein & ligandP + L PLkakdKa = [PL] = Association Constant [P][L]Kd = 1 = Dissociation Constant KaKa = =[P][L][PL] kakdThe fraction of occupied binding sites (θ) is proportional to the ligand concentrationP + L PLWhen [L] = Kd, then = 1/2Lower Kd = Higher Affinity!!Simple binding: Hyperbolic curveA protein with higher affinity for a ligand has a higher binding curve and lower KdAllosteric proteinBinding of a ligand (L1) to one site affects binding properties of ligand (L2) at another site (via a conformational change in the protein) .Modulator (L1) is an ‘activator’ if it increases affinity at 2nd site (where L2 binds)Modulator (L1) is an ‘inhibitor’ if it decreases affinity at 2nd site (where L2 binds)L1L2L1L2L LLLHeterotropic interaction:Modulator and other ligand are differentHomotropic interaction (cooperativity): Modulator and other ligand are the sameThe symmetry (concerted) model of cooperativitySubunits can adopt one of two possible conformations: T or R. All subunits must adopt the same conformation (protein is always symmetric). Equilibrium between T and R states is influenced by ligand or modulator binding. The sequential (gradual) model of cooperativity Subunits can adopt multiple conformations; Binding of ligand (S) induces conformational changes in the bound subunit and in neighboring subunits; Bound conformations may have higher or lower affinity for ligand than the free protein.The oxygen-binding curves of Mb and Hb reflect their different functionsMyoglobin: single subunit, high affinity to oxygen, hyperbolic curve.Hemoglobin: 4 subunits, sigmoidal curve, low affinity at tissues, high affinity at lungs. Cooperativitiy.•Heme is held in place by the proximal His and by hydrophobic residues•Proximal His of Hb covalently binds Fe of heme• Distal His hydrogen bonds to O2 bound to Fe. It reduces the affinity of hemoglobin to the toxic positive modulator CO by forcing CO to adopt an angle.Heme cofactors bind O2The proximal His links flattening of the heme to shifting of helix F in the T R transition.Movement of helix F shifts the entire quaternary structure of hemoglobinT-state = deoxygenated, low affinityR-state = oxygenated, high affinityThere are also several ion pairs in the T-state that are broken upon transition to the R-state.•Positive (stabilize R-state)–O2–CO (competitive inhibitor, P50 = 200x lower than O2 (would be 20,000x lower if distal His were not there)–NO–H2S•Negative (stabilize T-state)–2,3 BPG•Very negatively charged. Makes ionic interactions with Lys, Arg, His, N-terminus in center of tetramer. Keeps Hb in T-state •In R-state, conformation change closes up this central cavity and BPG cannot bind•At high altitudes, BPG helps transfer more oxygen to the tissues–H+ “Bohr Effect”•Protons help salt
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