title: Design of the helix-turn-helix motif: nonlocal effects of quaternary structure in DNA recognition investigated by laser Raman spectroscopy.
abstract: The operator-binding domain of phage lambda repressor provides a model for DNA recognition by the helix-turn-helix (HTH) motif. In the wild-type protein, dimerization is mediated by hydrophobic packing (of the dyad-related helix 5), which serves as an indirect determinant of operator affinity. The mutant repressor, Tyr88----Cys, forms an intersubunit disulfide linkage and exhibits enhancement of both structural stability and operator affinity. Yet the dimer-specific operator affinity of the mutant is 10-fold weaker than that of the wild-type (noncovalent) dimer, suggesting nonlocal effects of the intersubunit disulfide bond on HTH recognition (Sauer et al., 1986). To explore such nonlocal effects, we describe laser Raman studies of the Cys88 mutant repressor and its interaction with operator sites OL1 and OR3. The following results have been obtained: (i) Wild-type and mutant dimers exhibit similar secondary structures, indicated by quantitative comparison of Raman amide I and amide III bands. (ii) The engineered disulfide of the mutant lacks rigorous symmetry; we observe mainly the gauche/gauche/trans CC-S-S-CC rotamer. (iii) Remarkably, distinctive local and nonlocal differences are observed in the mechanisms of DNA recognition by wild-type and mutant repressors. These differences involve specific hydrogen-bonding interactions between the protein and DNA, including guanine N7 sites in the major groove of DNA, and alterations in DNA phosphodiester conformation induced by protein binding. We analyze these differences in relation to crystal structures of the wild-type dimer with and without bound DNA.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Division of Cell Biology and Biophysics, School of Basic Life Sciences, University of Missouri-Kansas City 64110.
authors.forename: J M
authors.initials: JM
authors.lastname: Benevides
authors.forename: M A
authors.initials: MA
authors.lastname: Weiss
authors.forename: G J
authors.initials: GJ
authors.lastname: Thomas
mesh_terms: D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins; D004220:Disulfides; D006860:Hydrogen Bonding; D007834:Lasers; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D009875:Operator Regions, Genetic; D011487:Protein Conformation; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics*; D012995:Solubility; D013059:Spectrum Analysis, Raman; D014443:Tyrosine / Q000737:chemistry; D014732:Vibration; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; D014961:X-Ray Diffraction
chemical_list: D004268:DNA-Binding Proteins; D004220:Disulfides; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D014443:Tyrosine; D004247:DNA