Context Dependence of Phenotype Prediction and Diversity in
Combinatorial Mutagenesis
Delagrave S., Goldman E. R., Youvan D. C.
Palo Alto Institute of Molecular Medicine, Mountain View, CA 94043, USA.
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 ß 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.