University:
Amherst College
Course:
CHEM 330 | Biochemical Principles of Life at the Molecular Level
Academic year:

2011
Views:

289

Pages:

14
Author:

Ronin Olson

1HHO structural information • Humans, 2.1 A • No expression system • By homologous chain: 1HGB, 1JEB, 1M9P Arg 141 Contacts in CSU Conservation Residue Conservation 1HHO Ramachandran plot Oxygen Binding Oxygen binding is cooperative. Oxygen binding changes the conformation of entire hemoglobin tetramer from T to R. Oxygen binds to the iron of the heme group of hemoglobin subunit. Consequently, the oxygen pulls up the iron which then also pulls up the histidine it has bonded to. This shift causes conformational changes in the single subunit, which then initiates changes in the other subunits to increase binding affinity. (This mechanism produces cooperative binding.) Sickle Cell Hemoglobin Mutation: Val replaces Glu at 6th position on each beta chain Val fits into hydrophobic pocket of the beta subunit of another hemoglobin tetramer, forming linear polymers. Blood cells become sickle-shaped (elongated) and cannot travel through capillaries, resulting in tissue death if blood cannot reach. Sickle cell anemia (hemoglobin S) protects against malaria: sickle cells break down and are removed by the spleen more readily when infected with Malaria parasite Voet, 195-197 Fetal Hemoglobin Fetal hemoglobin has higher affinity for oxygen than maternal hemoglobin, making transfer of oxygen from mother’s blood more successful. Conformation: fetal hemoglobin has gamma chains instead of beta and instead of His 143 (cation), has Ser 143 (uncharged) His usually stablizes deoxyhemoglobin, so if replaced by Ser, infant hemogloblin destabilizes deoxyhemoglobin, thereby stabilizing oxyhemoglobin. Hemoglobin Structure: O2 Binding Oxy R state relaxed Deoxy T state tense Hb + 4O2 ⇌ Hb(O2)4 * Do these data support the statement that Hb is a dimer of dimers? * What about the structural change from deoxy to oxy? Hb: Changes at Heme upon O2 Binding •T state Lighter lines are deoxy Hb, Fe out of plane •R state Bold Lines are oxy Hb, Fe in plane of Heme Hb + 4O2 ⇌ Hb(O2)4 Protein Function Cooperative Binding of Ligands • examine 2 ligands binding in concert (binding of 1st ligand immediately followed by binding of 2nd ligand) – Po + L ⇆ P1 Kd1 = [Po][L]/[P1] first ligand – P1 + L ⇆ P2 Kd2 = [P1][L]/[P2] second ligand • Net reaction: – Po + 2L ⇄ P2 – Po = (1-Y)(PT) Kd1Kd2 = Po[L]2/P2 – Plot of ln(Y/1-Y) vs ln[L] will give – slope of n, the number of sites [ Po ][ L] 2 K1 K 2  P2 K 1 K 2 P2  [(1  Y ) PT ][ L] 2 P2 [ L] 2  [(1  Y ) PT K 1 K 2 Y [ L] 2  1  Y K1 K 2  Y  ln    2 ln[ L]  ln( K 1 K 2 ) 1 Y  Protein Function Cooperative Binding of Ligands • Now, consider a protein with 4 cooperative sites • • • • Po + L ⇆ P1 P1 + L ⇆ P2 ……… P3+ L ⇆ PL4 K1d = Po[L]/P1 K2d = P1[L]/P2 • • • • • • How many individual microstates exist? 4 infinitely cooperative sites, a,b,c,d: Po = Po ; P4=Pabcd 1 microstate P1 = Pa, Pb, Pc, Pd 4 microstates P2 = Pab, Pac, Pad, Pbc, Pbd, Pcd 6 microstates P3 = Pabc, Pbcd, Pabd, Pacd 4 microstates At limit, perfect cooperativity, system doesn’t exist in intermediate states K4d = P3[L]/P4 Kdoverall = Po[L]4/P4 • Again, above are macroscopic dissociation constants……. Protein Function Cooperative Binding of Ligands Hb has four binding sites Unlike Mb or Tf, L binding to Hb is not hyperbolic but sigmoidal binding of first ligand increases the affinity of the remaining sites for ligand Examine concerted model: Hb + 4O2 ⇌ Hb(O2)4 Ka Hb(O2)4 ⇌ Hb + 4O2 Kd Protein Function Cooperative Binding of Ligands O2 binding is cooperative Hb + 4O2 ⇌ Hb(O2)4 Ka Hb(O2)4 ⇌ Hb + 4O2 Kd Kd = [Hb][O2]4/[Hb(O2)4] Y = [Hb(O2)4]/[HbT]] Y = pO2n/ Kd+ pO2n if perfect cooperativity, n = 4

BioChem 330 Outline: Bio-molecular Structure Function (Part 2)

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