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An algorithmically optimized combinatorial library screened by digital imaging spectroscopy.

E R Goldman·D C Youvan
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Abstract

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.

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AlgorithmsAmino Acid SequenceBacteriochlorophyllsBase SequenceBinding SitesDNA, BacterialEscherichia coliGene LibraryImage Processing, Computer-AssistedLight-Harvesting Protein ComplexesMolecular Sequence DataMutagenesisMutagenesis, InsertionalPhotosynthetic Reaction Center Complex ProteinsPhylogenyPlasmidsRhodobacter capsulatusSpectrum AnalysisComparative StudyJournal ArticleResearch Support, U.S. Gov't, Non-P.H.S.Research Support, U.S. Gov't, P.H.S.

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E R Goldman, D C Youvan. 1992. An algorithmically optimized combinatorial library screened by digital imaging spectroscopy.. https://doi.org/10.1038/nbt1292-1557

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