COMPARISON OF PROTEIN STRUCTURES IN SOLUTION USING LOCAL CONFORMATIONS DERIVED FROM NMR DATA - APPLICATION TO CYTOCHROME-C

Structural comparisons of proteins in solution are often required to e xamine structure-functional relationships, study structural effects of mutations or distinguish between various forms of the same molecule u nder different conditions. A nuclear magnetic resonance (NMR) based pr obabilistic strategy is presented and used to study the structural dif ferences between the two redox states of cytochrome c in solution. A p robabilistic approach is employed to calculate the main chain conforma tions of horse ferro- and ferricytochrome c in solution, based on the published sequential d connectivity data. Conformational differences b etween the two oxidation states of horse cytochrome c in solution are found to be statistically significant The largest changes in conformat ion are at residues Lys27, Thr28, Leu32, Gln42, Thr47, Tyr48, Thr49, G lu69, Lys72, Met80, Phe82, Ile85 and Lys86, all of which are close to the heme (within 14 Angstrom of the heme iron in the high resolution X ray structure of tuna cytochrome c). We suggest that these conformatio nal changes may modulate local dipole moments and hence influence the interactions of cytochrome c with its physiological redox partners dur ing the electron transfer process. The oxidation state dependent confo rmational differences are found to be much greater in solution than in the crystalline state, and the solution and crystal structures differ significantly in regions close to the heme. These results suggest tha t the highly charged nature of cytochrome c makes this protein particu larly sensitive to the ionic strength of its environment and leads to differences between crystal and solution structures in the same oxidat ion state. In such cases, crystal structures must be used with caution for modeling molecular interactions in vivo. More generally, this ana lysis indicates that the determination of accurate local conformations based on nmr data can provide useful information about structure-func tional aspects of proteins in solution.