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

Abstract

Site-directed mutagenesis and combinatorial libraries are powerful tools for providing information about the relationship between protein sequence and structure. Here we report two extensions that expand the utility of combinatorial mutagenesis for the quantitative assessment of hypotheses about the determinants of protein structure. First, we 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 ␣-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. Site-directed mutagenesis (1) and combinatorial libraries (2-19) have been used to generate considerable information about the structure-determining elements in proteins. The fraction of variants in combinatorial libraries containing randomized residues (2-8) or residues constrained to be hydrophobic (9-15) that pass some test have been used as semiquantitative assessments of the role of the targeted residues or motifs. A more hypothesis-oriented approach used degenerate codons to construct binary or hydrophobic-hydrophilic patterns in library variants whose effects on protein structure could then be tested (16)(17)(18)(19). However, only a limited number of types of hypotheses can be tested if one uses degenerate codons (codons with mixed nucleotides at one or more sites) to introduce variability into a library. We report here two tools to extend the range of hypotheses that can be quantitatively assessed with combinatorial libraries.The first extension involves the use of resin-splitting technology (20) to facilitate the construction of arbitrarily complex libraries that are free of the constraints imposed by the genetic code. Libraries can be constructed so that all of their members conform to some hypothesis, and members can then be scored by some structurally related test. As in previous applications, the successful fraction of variants serves as a relative ''score'' of the hypothesis, but the arbitrarily complex nature of the hypotheses made possible by split-resin technology extends the range of what can be tested.The second extension involves the use of regression analysis to expand the analytical power of combinatorial library experiments. In an ideal case, one would be able to assess a hypothesis concerning the determinants of protein structure by making a small number of carefully chosen mu...

Breaking open a protein barrel

“…Many other factors are thought to influence the folding of a protein. The role of local structure and identity of side chains in helical regions has been considered important: Gly and Pro, for example, destabilize helical structure, whereas Ala tends to favor helix (20,21). Signals at the ends of short helices also have been implicated: these include capping structures, in which polar side chains with the backbone near the ends of a helix (22).…”

Lysozyme among the Lilliputians

“…5) were a large step in the right direction, but a gap remains between current methods and the full repertoire of site-diversity and combinatorial complexity that is ultimately desired. That gap has now been filled by Lahr et al (2).…”

“…Now comes the sequel-patterned library analysis-in another landmark paper, reported in a recent issue of PNAS by Lahr et al (2), and closed the millennium on a high note. But what is patterned library analysis?…”

“…To validate their approach, Lahr et al (2) assessed helix propensities in eglin c, a 70-residue proteinase inhibitor with a solitary helix. A close eglin homologue, CI2, has been utilized extensively in folding studies (6).…”

“…The method uses resin-splitting technology to create libraries of arbitrary complexity by generating mutants at any number of residue positions, with each position varied according to its own menu of residue choices. For example, in the sequence below, four residue positions (2,5,7, and i) are selected for mutation. Each position is varied within a different list of residue options (lists 1, 2, 3, and 4, with n 1 , n 2 , n 3 , and n 4 members, respectively), resulting in a library with n 1 ϫ n 2 ϫ n 3 ϫ n 4 ϭ N possible transformants.…”