This proposal addresses two types of problems relevant to heme-containing proteins: general questions on the relationship between primary sequence and secondary and tertiary structure, and specific questions on the role of the prosthetic group in determining the structure and stability of the native material. The heme-containing hydrophilic domain of hepatic cytochrome b5 (cyt b5) is chosen as model system. Much is known about cyt b5 function and structure. Yet the nature and relative importance of the interactions between the prosthetic group and the protein matrix are not fully understood, and the structure dictated by the polypeptide chain alone has not been characterized in detail. Cyt b5 will be subjected to systematic studies aimed at defining the various interactions. (a)The apoprotein of cyt b5 will be investigated in detail by circular dichroic and proton nuclear magnetic resonance spectroscopy. The existing elements of secondary structure will be identified and the way in which they pack against each other described; the stability of the structure will be tested by varying external conditions; the kinetics of heme binding and its conformational consequences will be monitored. (b)Apocyt b5 will be reconstituted with protoporphyrin IX, i.e. the prosthetic group without the central iron atom. Structural data will be obtained on the """"""""des-Fe"""""""" protein to extract the interactions between the protein and the protoporphyrin ring. (c)Single- and double-site mutant proteins will be produced and their apoprotein probed by the same methods as applied to the wild-type apoprotein. The mutants will reveal the contribution of selected amino acids to the structure of the apoprotein and its stability. The data collected in (a), (b), and (c) will be compared to the corresponding information available on the reduced and oxidized forms of the holoprotein. Thus the roles of amino acid sequence, prosthetic group, and iron atom in fixing the native holoprotein conformation and determining its thermodynamic properties will be characterized individually. The conclusions of this work will apply to a better understanding of the structural determinants of proteins.
