| 302Combinatorial libraries of alanine-substituted proteins can beused to rapidly identify residues important for protein function,stability and shape. Each alanine substitution examines thecontribution of an individual amino acid sidechain to thefunctionality of the protein. The recently described method ofshotgun scanning uses phage-displayed libraries of alanine-substituted proteins for high-throughput analysis.AddressesDepartment of Chemistry, University of California, Irvine,CA92697-2025, USA*e-mail: gweiss@uci.eduCurrent Opinion in Chemical Biology2001, 5:302–3071367-5931/01/$ — see front matter? 2001 Elsevier Science Ltd. All rights reserved.AbbreviationshGHhuman growth hormonehGHbphGH-binding proteinIntroductionCombinatorial protein libraries offer powerful tools to dissect biological phenomena. Protein libraries can be usedto explore the intricate relationship between the primaryamino acid sequence and protein shape, stability and activity. Mutagenesis of specific residues in proteins hasproven invaluable in probing the contributions of individ-ual amino acid sidechains to the properties of proteins.The seminal chemical syntheses of proteins with modifiedsidechains by Merrifield and co-workers (for a representa-tive example, see [1]), for example, elucidated theenzymatic activity of RNase A. Fersht, Winter andco-workers [2] elegantly demonstrated the power ofoligonucleotide-directed mutagenesis to measure hydrogen-bond strengths in proteins. Combinatorial libraries of mutant proteins can be used torapidly analyze protein function and identify importantsidechain functionalities. For the examination of recep-tor–ligand interactions, protein libraries can be generatedwith either random [3,4,5?] or site-specific mutations.Saturation mutagenesis substitutes specific positions withall 20 naturally occurring amino acids [6,7]. However, sub-stitution of a few carefully chosen amino acids can providea thorough portrait of protein function and simplify analy-sis of the data. This review focuses upon combinatorialprotein libraries substituted with either alanine or thewild-type amino acid in specific positions. Wild type in thiscontext refers to leaving the residue unchanged as the naturally occurring amino acid sidechain. Because thistopic has not been reviewed before in this journal, the timeperiod reviewed here is longer than usual.Non-covalent binding interactions between receptors andligands are mediated by residues in direct contact, whichform a structural epitope. Biophysical techniques includingX-ray crystallography and multidimensional NMR revealsuch binding contacts. The structure alone, however, doesnot tell the whole story of how a protein works. Within thecontact area, a subset of residues may contribute the major-ity of binding energy, through hydrogen bonds, salt bridges,dipole–dipole interactions and hydrophobic interactions.Residues with energetically favorable contacts define thefunctional epitope [8]. A tightly clustered functional epitope resembling a cross section of protein structure (i.e.with hydrophobic center surrounded by hydrophilicCombinatori |
|
|
