PowerPoint PresentationSlide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Slide 25Slide 26Slide 27Slide 28Slide 29Slide 30Slide 31Slide 32Slide 33Slide 34Slide 35Slide 36Protein Function(Chapter 7)•O2 binding 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 + O2 CO2 + 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 -helical segments, A-H•His93 (proximal histidine) binds directly to iron •No other covalent attachments of heme to 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] Kd = [PL] = [L] [PL] + [P] [L] + Kd = fraction of the total binding sites occupied by ligand = [L] = fraction of the total binding [L] + Kd sites occupied by ligand For a gas, the concentration dissolved is proportional to the partial pressure of the gas over the solution. = pO2 pO2 + P50P50 = pO2 at 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% O2 bound98% O2 boundRectangularhyperbolaMb + O2 MbO2Mb has ~200x greater affinity for CO as compared to oxygenMyoglobin O2 Binding SiteDistal histidineProximal histidineFigure 7-5Both the and chains of Hb look like Myoglobin, but they differ in primary sequence.Hemoglobin (Hb) Carries Oxygen chain chainHb (2, 2)Oxygen binding siteHemoglobin (Hb) is an 22 tetramer •T state (tense) and R state (relaxed) represent two different conformations of the tetramer.•Both bind O2 but R state binds it more strongly•Interactions between 1 and 1 and between 2 and 2 are dominant and change little in the T-to-R conformational change.•The major shifts are at the interfaces between 1 and 2 (and 2 and 1)Figure 7-830 residuesform theinterfaces between and 19 residues for theinterface between (and ). and 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 relative to (2) Change at interface between and (3) R state is more compact(4) T state has additional salt bridges(5) In R state individual O2 sites have higher affinity for O2. - better Fe-O2 bond 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 (and ) interfaces.The trigger for the R to T state conversionHb binds oxygen cooperativelySigmoidal binding curveHill Plots are used to determine degree of cooperativityConcerted Sequential model model Two models for cooperative binding(H+)Hb + O2 HbO2 + H+Increases in [H+ ] or [CO2] cause decreased affinity for O2 and vice versaHb is regulated by H+ and CO2 The Bohr effect: the effect of pH and CO2 on the binding and release of oxygen to Hb.Lower pH (higher H+) stabilizes the T-state)lungstissueMore O2 releasedas 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 charge T-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 -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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