CONTEXT DEPENDENCE OF PHENOTYPE PREDICTION AND DIVERSITY IN COMBINATORIAL MUTAGENESIS

Two different combinatorial mutagenesis experiments on the light-harvesting II (LH2) protein of Rhodobacter capsulatus indicate that heuristic rules relating sequence directly to phenotype are dependent on which sets or groups of residues are mutated simultaneously, Previously reported combinatorial mutagenesis of this chromogenic protein (based on both phylogenetic and structural models) showed that substituting amino acids with large molar volumes at Gly(beta 31) caused the mutated protein 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 bacteriochlorophyll. In a second experiment described here, we have not used structural models or phylogeny in choosing mutagenesis sites, Instead, a set of six contiguous residues was selected for combinatorial mutagenesis. In this latter experiment, the residue substituted at Gly(beta 31) was not a determining factor in whether LH2 or LH1 spectra were obtained; therefore, we conclude that the heuristic rules for phenotype prediction are context dependent. While phenotype prediction is context dependent, the ability to identify elements of primary structure causing phenotype diversity appears not to be. This strengthens the argument for performing combinatorial mutagenesis with an arbitrary grouping of residues if structural models are unavailable.