Patterned library analysis: A method for the quantitative assessment of hypotheses concerning the determinants of protein structure

Site-directed mutagenesis and combinatorial libraries are powerful tools for providing information about the determinants of protein structure. I discuss the development of two methods that extend the effectiveness of libraries for providing information about the determinants of protein structure. First, I show that resin splitting technology, which allows the construction of arbitrarily complex libraries of degenerate oligonucleotides, can be used to construct more complex protein libraries for hypothesis testing than can be constructed from oligonucleotides limited to degenerate codons. Second, using eglin c as a model protein, we show that regression analysis of activity scores from library data can be used to assess the relative contributions to the specific activity of the amino acids that were varied in the library. The regression parameters derived from the analysis of a 455 member sample, from a library wherein four solvent-exposed sites in an alpha-helix can contain any of nine different amino acids, are highly correlated (P < 0.0001, R 2 ≥ 0.97) to the relative helix propensities for those amino acids, as estimated by a variety of biophysical and computational techniques.