Summary
The increasing sophistication of mutagenesis and screening technologies has led to the isolation of many biophysically important mutants of the photosynthetic reaction center and light harvesting antennae. Site-directed mutagenesis and structural motif rearrangements of these proteins have accelerated our understanding of the fundamental mechanisms of the light reactions of photosynthesis. Knowledge of structural requirements for pigment binding proteins has also been acquired through mutants. Combinatorial cassette mutagenesis promises to produce additional biophysically interesting mutants. Since protein structure and function can not yet be accurately predicted from protein sequence, this method has the ability to generate phenotypes that would not be found through point mutations or global rearrangements. Libraries generated through combinatorial cassette mutagenesis can be rapidly screened by digital imaging Spectroscopy, and mutants with interesting absorption spectra identified for further characterization. Currently, combinatorial cassette mutagenesis experiments have focused on the reaction center and light harvesting II antennae. Specific amino acid residues in the reaction center were randomly substituted, and the library subjected to a photosynthetic selection prior to screening. Using such methodology, mutants can be isolated which occur with a frequency of approximately one-in-a-million. In the light harvesting system, strategies for more efficiently searching sequence space are being explored. Methods have been implemented which limit the amino acids coded for at each mutated position. These methods enhance for the desired phenotype over random mutagenesis. Goals for mutants generated via combinatorial methods include searching for reaction centers that perform wrong way electron transfer, and redesigning light harvesting antennae to perform charge separation.