title: Contribution of a buried hydrogen bond to lambda repressor folding kinetics.
abstract: A hydrogen bond between the buried residues Asp 14 and Ser 77 in monomeric lambda repressor has been removed by mutation of these residues to alanine. Double mutant cycles show that the interaction stabilizes the native state of the protein by 1.5 kcal/mol. Removal of the interaction affects mainly the unfolding rates and not the folding rates, suggesting that this hydrogen bond is not substantially formed in the rate-limiting steps in the folding pathways of the protein. Mutations in two versions of lambda6-85, wild type and the faster folding G46A/G48A (WT), show similar effects. Diffusion-collision correctly predicts the behavior of WT but not of wild type. Our analysis suggests that folding of helix 3 is a crucial slow step along the various folding pathways and generally occurs before the formation of the 14-77 hydrogen bond. Experiments removing tertiary interactions, combined with experiments altering helical stability and diffusion-collision calculations, provide a strategy to unravel the folding mechanisms of small helical proteins.
authors.affiliation: Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: J K
authors.initials: JK
authors.lastname: Myers
authors.forename: T G
authors.initials: TG
authors.lastname: Oas
mesh_terms: D010582:Bacteriophage lambda / Q000737:chemistry* / Q000235:genetics; D004268:DNA-Binding Proteins; D006860:Hydrogen Bonding; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D054334:Viral Regulatory and Accessory Proteins
chemical_list: D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins