An algorithmically optimized combinatorial library screened by digital imaging spectroscopy.
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.
Goldman
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Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139.
Youvan
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D000465:Algorithms; D000595:Amino Acid Sequence; D001429:Bacteriochlorophylls / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D004269:DNA, Bacterial / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D007091:Image Processing, Computer-Assisted; D045342:Light-Harvesting Protein Complexes; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016254:Mutagenesis, Insertional; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry / Q000235:genetics*; D010802:Phylogeny; D010957:Plasmids; D016354:Rhodobacter capsulatus / Q000235:genetics*; D013057:Spectrum Analysis / Q000379:methods*
D001429:Bacteriochlorophylls; D004269:DNA, Bacterial; D045342:Light-Harvesting Protein Complexes; D045322:Photosynthetic Reaction Center Complex Proteins