The overall goal of this application is to identify the structural determinants of stability and dynamics in b type heme proteins that normally contain a tightly bound iron- porphyrin prosthetic group. Removal of the heme group from these proteins alters, but does not normally obliterate, the structure and stability of the overall tertiary structure of the polypeptide matrix. The resulting apoproteins provide a convenient system for investigating the structures of stable """"""""folding"""""""" intermediates which are neither native nor completely unfolded under physiological conditions. Previous work by Dr. Lecomte, which is described in the Progress Review, and by others, most notably the Baldwin and Wright groups, have indicated that single-domain apocytochrome b5 and apomyoglobin can be considered """"""""modular."""""""" One domain folds rapidly and is very stable in the absence of heme. The other is involved more directly in heme binding but is less stable and organized in the absence of the prosthetic group. This modular hypothesis will be tested by new and more detailed NMR studies of native and mutant soluble fragments of rat liver apocytochrome b5 and recombinant sperm whale apomyoglobins. Five specific projects are described. (1) The temperature response of the structure and dynamics of apocytochrome b5 will be examined by heteronuclear NMR techniques. A refined description will be sought to determine how closely the structure of the stable N and C terminal module resembles that in the holoprotein and the extent of unfolding of the internal heme binding domain when the prosthetic group is removed. (2) Site-directed mutagenesis will be used to probe specific elements of secondary structure. In particular, the pH dependence of end capping and the role of tertiary context in stabilizing helices 1 and 6, the N and C terminal regions, respectively, will be studied. (3) A truncated model of the stable module in cytochrome b5 will be constructed by excising the middle of the DNA sequence (corresponding to residues 44-71). Previous NMR results suggest that this region should form a stable module similar to that observed in the intact apoprotein. If this proves to be true, then the modular picture for cytochrome b5 folding will be firmly established. The second module can then be added back in stages to investigate its role in conferring stability and forming the heme binding site. (4) 3D NMR techniques will be used to determine the main structural and dynamic features of sperm whale apomyoglobin in solution. As in the case of apocytochrome b5, a well-organized core coexists with more disordered regions, and site-directed mutagenesis can be used to probe individual regions of secondary and tertiary structure. (5) A truncated version of apomyoglobin will be constructed using the sequence of the central exon in the mammalian gene. A similar minimyoglobin has been made using limited proteolysis by Brunori and coworkers in Rome, demonstrating the feasibility of this approach. Again, the idea is to see if this exon codes for a stable module in apomyoglobin.
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