FBICH 410 1st Edition Lecture 12Outline of Last Lecture - Protein SequencingOutline of Current Lecture - Myoglobin and hemoglobin- Protein ligand bindingo Ligands can be proteins, small organic or inorganic compounds, and bind noncatalyticallycausing conformational changes and can be regulatory- Myoglobin (Mb)- O2 storage and transport in muscles- monomero 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 coloro 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 hemeo CO binds tighter than O2- If Mb bound heme, then CO Kd is only 200x smaller than O2o 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 liquido 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 spotsThese 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 O2o 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 increaseo 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 cappilarieso 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 stateo No O2 around and the T state predominates to conformation of deoxyhemoglobin- Structure of Hb- alpha2beta2 tetramer- dimer of dimer alpha betao A1b1 and a2b2 function as a protomerso Alpha-beta dimer associate via ionic interactions and H bondso 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 2alpha-beta 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 cooperativityo 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 4o N=1 noncooperative binding- hyperbolic curveo N>1 positive cooperativeo N<1 negative cooperative- Hill Plot Y/(Y-1)=[pO2]n /[p50]no N=slope of lineo Plot log[y/1-y] vs.