F BICH 410 1st Edition Lecture 12 Outline of Last Lecture Protein Sequencing Outline of Current Lecture Myoglobin and hemoglobin Protein ligand binding o Ligands can be proteins small organic or inorganic compounds and bind noncatalytically causing conformational changes and can be regulatory Myoglobin Mb O2 storage and transport in muscles monomer o Sphereical and 90 composed of alpha helicies Hemoglobin Hb 02 storage and transport in blood tetramer Hb and Mb were first crytal structure that was solved Oxygen binds to heme complex O2 not very soluble in aqueous solutions so binds to carrier to increase solubility Amino acids don t reversibly bind 02 but transition metals do Cu or Fe Fe incorporated into a prosthetic group heme can be used to transport O2 02 binding to heme in Mb FeII is coordinated by 4 porphyin N atoms and 1 N from His F8 proximal His When o2 binds acts as 6th ligand to FeII His E7 distal His His 64 7th residue of Ehelix H bonds to 02 Oxygenation of Heme and color of blood the binding of 02 to FE heme complex alters the electronic state of changing color o Purple venous blood red arterial blood Can use absorbance to calculate oxygenated to deoxygenated hemoglobin Oxidation of FeII to FeIII prevents O2 binging FeIII is brown colored and known as methemoglobin CO2 NO H2S higher binding affinity than O2 Dissociation constant Kd for CO is 20000x smaller than Kd of 02 for unbound heme o CO binds tighter than O2 If Mb bound heme then CO Kd is only 200x smaller than O2 o Because of steric interference with distal His His E7 Binding of O2 to Mb varies with 02 pressure Kd Mb pO2 MbO2 Henry s law quantity of gas absorbed by a liquid is proportional to partial pressure of gas above liquid o Use Kd to determine affinity of protein for a ligand Fractional percent of 02 binding is also known as percent oxygen saturation Y binding sites occupied total of binding spots These notes represent a detailed interpretation of the professor s lecture GradeBuddy is best used as a supplement to your own notes not as a substitute Y pO2 Kd pO2 Y pO2 p50 pO2 Affinity constant p50 kD oxygen pressure at which Mb 50 saturated w O2 o When Kd pO2 50 of binding spots occupied High p50 LOW AFFINITY low p50 high affinity When O2 binds to Mb binding increase is hyperbolic with pressure increase o Binding weaker if it takes a greater pO2 to get p50 Hemoglobin sigmoidal graph structure Hb blood delivers to Mb muscle therefore must pick up and deliver to tissue and have the ability to release O2 in cappilaries o Mb has lower p50 and higher affinity Hb has 2 stable states T tension and R relaxed o O2 can bind to both but has higher affinity for R state o No O2 around and the T state predominates to conformation of deoxyhemoglobin Structure of Hb alpha2beta2 tetramer dimer of dimer alpha beta o A1b1 and a2b2 function as a protomers o Alpha beta dimer associate via ionic interactions and H bonds o Changes of pH effect more of the sliding contacts of dimer dimer because of hydrophobic interactions not effected so alpha beta dimer not affected T and R transition stabilized by series of ion pairs that loe at the omtersectopm between 2alphabeta protomers O2 binding initiates series of movements resulting in T to R shift Movement affect quarternary structure O2 biding causes conformation change to occur at heme o Without O2 Fe is out of heme plane with O2 Fe is in heme plane If iron moves His F8 move causing F helix to move causing FG corner to move In T state heme of beta subunits are inaccessible to O2 O2 binding causes conformational change in both Mb and Hb As more O2 binds transitions from low affinity T hyperbolic to high affinity sigmoidal this is called positive cooperativity o As one binds it is easier for the next one to bind Hb Y pO2n p50n pO2n o N hill coefficients cant be greater than 4 o N 1 noncooperative binding hyperbolic curve o N 1 positive cooperative o N 1 negative cooperative Hill Plot Y Y 1 pO2 n p50 n o N slope of line o Plot log y 1 y vs logpO2