The research described in this proposal is aimed at understanding the interrelationships of structure and function in a multi-subunit complex of membrane proteins. An important emphasis in the proposed research is based on """"""""directed molecular evolution""""""""; in this approach a protein domain is randomly modified, and subsequently the protein complex is selected for a particular functional property. For this purpose, the membrane-protein complex of photosystem II in the cyanobacterium Synechocystis sp. PCC 6803 is utilized. The photosystem II complex has been chosen as an appropriate membrane-protein complex for this study because the approximate topology and presumed folding of the polypeptides are reasonably well understood and various domains of particular physiological importance are known. Synechocystis sp. PCC 6803 is a naturally transformable organism, DNA can be integrated into its genome by homologous recombination, and the cyanobacterium can be propagated in the absence of functional photosystem II if glucose is provided (photoheterotrophic growth). In this system, domains of a photosystem II gene (psbA, coding for the D1 protein) will be replaced by degenerate DNA sequences. Subsequently, using specific and strong selection pressure related to photosystem II function, a particular phenotype will be selected. The sequence of the altered psbA gene will then be determined. In this way, information on the structural and functional requirements of the protein domain will be obtained. In addition, naturally derived pseudorevertants with physiologically active photosystem II will be generated from genetically engineered mutants that carry small deletions in psbA; these deletions lead to a loss of photosystem II function, and pseudorevertants will be selected for restoration of this function. Upon examination of the sequence of the modified gene in these pseudorevertants, detailed information will be obtained regarding the requirements of the protein sequence in the modified region with respect to the physiology of the organism. Using both the """"""""directed molecular evolution"""""""" and pseudorevertant approaches, important functional features of the protein can be easily analyzed in terms of the degree of structural variability allowed. The results of this research are expected to contribute to an understanding of the role and structural flexibility of domains in the membrane protein studied. Even though membrane-protein complexes are ubiquitous in nature, and often fulfill crucial roles in cell physiology, little is understood regarding factors influencing folding, structure, and function of such complexes. The work described in this proposal is aimed towards increasing this understanding.
