An Algorithmically Optimized Combinatorial Library Screened by Digital
Imaging Spectroscopy
Goldman E.R., Youvan D.C.
Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139.
Combinatorial cassettes based on a phylogenetic "target set" were used to
simultaneously mutagenize seven amino acid residues on one face of a transmembrane alpha
helix comprising a bacteriochlorophyll binding site in the light harvesting II antenna of
Rhodobacter capsulatus. This pigmented protein provides a model system for developing
complex mutagenesis schemes, because simple absorption spectroscopy can be used to assay
protein expression, structure, and function. Colony screening by Digital Imaging
Spectroscopy showed that 6% of the optimized library bound bacteriochlorophyll in two
distinct spectroscopic classes. This is approximately 200 times the throughput (ca. 0.03%)
of conventional combinatorial cassette mutagenesis using [NN(G/C)]. "Doping"
algorithms evaluated in this model system are generally applicable and should enable
simultaneous mutagenesis at more positions in a protein than currently possible, or
alternatively, decrease the screening size of combinatorial libraries.