• Structural and dynamic properties of hemeproteins that control electroreactivity

      Dangi, Bindi A.; Guiles, Ronald (1999)
      The main objective of this project was to gain a better understanding of the structural and dynamic contributors to hemeprotein electroreactivity. To this end we employed cytochrome b5 as a model system. We have been successful in structurally characterizing cytochrome b5 in both the A and the B conformations which differ in the orientation of the bound heme. This was the first complete structural characterization of such a heterogeneous system. Differences in the structures have revealed the possible origins of differences in the reduction potential of the two conformers. Also, the dynamic behavior of cytochrome b5 was analyzed in the oxidized and reduced state. The results of this analysis have shown that changes in backbone dynamics of cytochrome b5 on oxidation contribute significantly to the electromotive driving force and that the dramatic difference in the dynamics of the two oxidation states are probably a result of redox linked hydrogen bond strength modulation. The observations have opened new avenues in terms of strategies that can be used for the development of therapeutic agents for the treatment of methemoglobinemia. A generalized hydrophobic binding domain on cytochrome b5 has been identified using an experimental NMR based strategy and binding to this domain could be further optimized to give a potential drug. Preliminary evidence based on the temperature dependence of the reduction potential suggests that the entropic changes associated with oxidation of myoglobin and hemoglobin are diametrically opposite to the behavior observed for cytochrome b5. An analysis of the backbone dynamics of myoglobin would give us insight into whether the backbone dynamics of a protein determine the electrochemical properties essential to the physiological role of a hemeprotein. In order to test this hypothesis, we have developed a high yield overexpression system for sperm whale myoglobin so as to obtain large quantities of the protein for NMR experiments. Further, a high yield expression system for the beta-globin subunit of hemoglobin has also been developed, the motivation being the study of the cytochrome b5-hemoglobin complex and the development of suitable remedial agents for sickle cell anemia.
    • Structural and electrochemical properties of wild-type and mutant cytochromes b-(5)

      Sarma, Siddhartha P.; Guiles, Ronald (1996)
      The main objective of this thesis project was to gain a better understanding of the protein structural factors that modulate heme reduction potentials in b-type cytochromes. Rat liver microsomal cytochrome {dollar}b\sb5{dollar} was chosen as a model system because the availability of a synthetic gene coding for the protein permits mutagenic experiments to be performed. The synthetic gene encoding for cytochrome {dollar}b\sb5{dollar} was subcloned into a high yield bacterial overexpression system that enabled isotopic enrichment of proteins. Site directed mutants of cytochrome {dollar}b\sb5{dollar} were prepared by subcloning the synthetic gene into bacteriophage DNA m 13mp18. Mutants of cytochrome {dollar}b\sb5{dollar} were designed to achieve reorientation of individual axial imidazole ligands. The orientation of the axial ligand planes is thought to modulate the reduction potential of bis(imidazole) axially ligated heme proteins. The A67V (alanine to valine) mutation resulted in a reorientation of the H63 imidazole ring and a shift in reduction potential by {dollar}-20{dollar} mV. Structural characterization of the A67V mutant protein was achieved using homonuclear and heteronuclear NMR methods. Calculation of the orientation of the components of paramagnetic susceptibility tensor showed that the wild type and mutant proteins differed only in the orientation of the z-component. The rotation of the z-component of the susceptibility tensor is in the same direction as the rotation of the H63 imidazole ring. EPR and Near-IR data suggest that the stability of iron d-orbital energy levels in the reduced proteins may be significant in determining the reduction potential of b-type heme proteins. Multidimensional double and triple resonance NMR methods have been applied to assign the backbone and side-chain {dollar}\sp{lcub}13{rcub}{dollar}C resonances for both equilibrium conformers of ferricytochrome {dollar}b\sb5{dollar} On the basis observed NOEs and backbone {dollar}\sp{lcub}13{rcub}{dollar}C chemical shifts, the solution secondary structure of cytochrome {dollar}b\sb5{dollar} has been determined. The {dollar}\sp{lcub}13{rcub}{dollar}C chemical shifts of backbone and side-chain atoms are relatively insensitive to paramagnetic effects. The reliability of such methods in anisotropic paramagnetic systems, where large pseudocontact shifts are observed, is evaluated through calculations of the magnitude of such shifts.