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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM051556-01
Application #
2190165
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1994-12-01
Project End
1998-11-30
Budget Start
1994-12-01
Budget End
1995-11-30
Support Year
1
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Arizona State University-Tempe Campus
Department
Other Basic Sciences
Type
Schools of Arts and Sciences
DUNS #
188435911
City
Tempe
State
AZ
Country
United States
Zip Code
85287
Keilty, A T; Vavilin, D V; Vermaas, W F (2001) Functional analysis of combinatorial mutants with changes in the C-terminus of the CD loop of the D2 protein in photosystem II of Synechocystis sp. PCC 6803. Biochemistry 40:4131-9
Vavilin, D V; Vermaas, W F (2000) Mutations in the CD-loop region of the D2 protein in Synechocystis sp. PCC 6803 modify charge recombination pathways in photosystem II in vivo. Biochemistry 39:14831-8
Keilty, A T; Ermakova-Gerdes, S Y; Vermaas, W F (2000) Probing the CD lumenal loop region of the D2 protein of photosystem II in Synechocystis sp. strain PCC 6803 by combinatorial mutagenesis. J Bacteriol 182:2453-60
Vavilin, D V; Ermakova-Gerdes, S Y; Keilty, A T et al. (1999) Tryptophan at position 181 of the D2 protein of photosystem II confers quenching of variable fluorescence of chlorophyll: implications for the mechanism of energy-dependent quenching. Biochemistry 38:14690-6
Howitt, C A; Vermaas, W F (1998) Quinol and cytochrome oxidases in the cyanobacterium Synechocystis sp. PCC 6803. Biochemistry 37:17944-51
Vermaas, W F (1998) Gene modifications and mutation mapping to study the function of photosystem II. Methods Enzymol 297:293-310
Kless, H; Vermaas, W (1996) Combinatorial mutagenesis and structural simulations in the environment of the redox-active tyrosine YZ of photosystem II. Biochemistry 35:16458-64
Kless, H; Vermaas, W (1995) Tandem sequence duplications functionally complement deletions in the D1 protein of photosystem II. J Biol Chem 270:16536-41