CONTEXT DEPENDENCE OF PHENOTYPE PREDICTION AND DIVERSITY IN COMBINATORIAL MUTAGENESIS

Two different combinatorial mutagenesis experiments on the light-harve sting II (LH2) protein of Rhodobacter capsulatus indicate that heurist ic rules relating sequence directly to phenotype are dependent on whic h sets or groups of residues are mutated simultaneously, Previously re ported combinatorial mutagenesis of this chromogenic protein (based on both phylogenetic and structural models) showed that substituting ami no acids with large molar volumes at Gly(beta 31) caused the mutated p rotein to have a spectrum characteristic of light-harvesting I (LH1). The six residues that underwent combinatorial mutagenesis were modeled to lie on one side of a transmembrane alpha-helix that binds bacterio chlorophyll. In a second experiment described here, we have not used s tructural models or phylogeny in choosing mutagenesis sites, Instead, a set of six contiguous residues was selected for combinatorial mutage nesis. In this latter experiment, the residue substituted at Gly(beta 31) was not a determining factor in whether LH2 or LH1 spectra were ob tained; therefore, we conclude that the heuristic rules for phenotype prediction are context dependent. While phenotype prediction is contex t dependent, the ability to identify elements of primary structure cau sing phenotype diversity appears not to be. This strengthens the argum ent for performing combinatorial mutagenesis with an arbitrary groupin g of residues if structural models are unavailable.