Protein Function(Chapter 7)•O2binding and transport:hemoglobin and myoglobinA classic model of protein function•Immune system: immunoglobulin One of the most abundant proteins in the blood•Muscle action: actin and myosinLungspO2~ 13kPaTissuepO2~ 4kPa~34% by weight hemoglobin (Hb)Red blood cells(erythrocytes)(Myoglobin)O2O2Glucose + O2CO2+ Water+ Useful Energy (ATP)Porphyrin molecules:•Tetrapyrrole macrocyclic compounds •Bind metal ions readily in center, especially ironHeme is a prosthetic group, a group that is permanentlyassociated with a protein’s native structure and function.Heme consists of a protoporphyrin ring bound to ferrousiron ( Fe2+). The iron has six coordination bonds, fourto nitrogens within the ring andtwo perpendicular to the ring.Iron protoporphyrin IX or “heme”Propionic acid groupsHydrophobic faceHydrophilic faceVinyl groupsNNNNHisO2Myoglobin, “Mb”•153 amino acid residues•eight α-helical segments, A-H•His93 (proximal histidine) binds directly to iron•No other covalent attachments of hemeto protein (contrast cytochrome c)Oxygen can only bindto one side of theprotein protected heme.When oxygen binds, the electronic propertiesof heme-iron change,turning from darkpurple to bright redPL P + LProtein = PLigand = LProtein-ligand complex = PLDissociation constant = Kd(= 1/Ka)Kd = [P][L] or [PL] = [P][L] [PL] Kdθ = [PL] = [L] [PL] + [P] [L] + Kd= fraction of the total binding sites occupied by ligandθ = [L] = fraction of the total binding [L] + Kdsites occupied by ligandFor a gas, the concentration dissolved is proportional to the partial pressure of the gas over the solution.θ = pO2pO2+ P50P50= pO2at half saturation, i.e., [Mb] = [MbO2]Myoglobin (Mb) oxygen binding curve Figure 7-4btissuelungMb is not a good oxygen transporter.4 / ( 0.26 + 4) =13 / ( 13 + 0.26) =94% O2bound98% O2boundRectangularhyperbolaMb + O2MbO2Mb has ~200x greater affinity for CO as compared to oxygenMyoglobin O2Binding SiteDistal histidineProximal histidineFigure 7-5Both the α and β chains of Hb look like Myoglobin, but they differ in primary sequence.Hemoglobin (Hb) Carries Oxygenα chainβ chainHb (α2, β2)Oxygen binding siteHemoglobin (Hb) is an α2β2tetramer•T state (tense) and R state (relaxed) represent two different conformations of the tetramer.•Both bind O2but R state binds it more strongly•Interactions between α1and β1and between α2and β2are dominant and change little in the T-to-R conformational change.•The major shifts are at the interfaces between α1 and β2(and α2 and β1)Figure 7-830 residuesform theinterfaces betweenα1β1 (and α2β2).19 residues for theinterface between α1β2 (and α2β1).α1β1 and α2β2 are the strongest interfacesStructure of HemoglobinIon pairs that stabilize the T state of deoxyhemoglobinInteractions that stabilize the T-stateLow Affinity T stateHigh Afinity R StateT vs R State(1) 15 degree rotation of α1β1 relative to α2β2(2) Change at interface between β1α2 and β2α1(3) R state is more compact(4) T state has additional salt bridges(5) In R state individual O2sites have higheraffinity for O2. - better Fe-O2bond length- fewer steric repulsionsPuckered and Fe out of planeFe moves in plane, pulling proximal His and Helix FHelix F is moved as oxygen binds. This promotes subunit rotation and rearrangementof the α1β2 (and β2α1) interfaces.The trigger for the R to T state conversionHb binds oxygen cooperativelySigmoidal binding curveHill Plots are used to determine degree of cooperativityConcerted Sequential modelmodelTwo models for cooperative binding(H+)Hb + O2 HbO2+ H+Increases in [H+ ] or [CO2] cause decreased affinity for O2and vice versaHb is regulated by H+ and CO2The Bohr effect: the effect of pH and CO2on the binding and release of oxygen to Hb.Lower pH (higher H+) stabilizes the T-state)lungstissueMore O2releasedas the pH is loweredProtonation of His HC3 in T state is a major contributor to Bohr effectCO2(high in tissues) is also carried by hemoglobinThis reaction produces protons and additional salt bridges stabilize the T state.BPG binding stabilizes the T-state of deoxyHbBPG in blood normally 5 mM, but it rises at high altitidesBlue indicates area of high positive chargeT-state R-stateBinding pocket open Binding pocket closedBPG binding stabilizes the T-state of deoxyHbSickle-Cell AnemiaThere are over 300 variants of Hb. 95% of the variants differ by only a single amino acid in the primary sequence of either the alpha or beta chain of Hb.Sickle-cell anemia is caused by a mutant form of hemoglobin, called HbS. HbS contains a single Glu to Val change at amino acid number six in the beta chain. 10% of African Americans are heterozygotic for HbS.Subtle difference in surface charge in beta chain creates a hydrophobicpatch in the HbS protein.The substitution of Val for Glu at postion 6 in the two β-chains causes the deoxyhemoglobin to lose its solubility and consequently aggregate and form fibersProtein AggregationSickle-Cell Anemia is a Molecular Disease of
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