title: Contribution of hydrogen bonding to protein stability estimated from isotope effects.
abstract: An unresolved issue in structural biology concerns the relative contribution of H bonds to protein stability. We use the small molecules 4-acetamidobenzoic acid and N-acetylanthranilic acid as model compounds to relate the energetic contribution from hydrogen bonds (H bonds) to the deuterium/hydrogen amide isotope effect. N-Acetylanthranilic acid models carbonyl-amide H bonds formed during protein folding; 4-acetamidobenzoic acid models the unfolded state in which the amide H bonds to water. NMR is used to measure shifts in the pK(a) of the ionizable carboxyl group when the amides of the compounds are either protonated or deuterated. From the pK(a) shift, we obtain a quantitative scale factor: SF = partial partial differential(DeltaG(HB))/partial partial differential(RT ln Phi), where DeltaG(HB) is the change in free energy of an H bond upon isotope substitution and Phi is the fractionation factor. Isotope effect data also are reported for a small globular protein, lambda repressor, using the "C(m) experiment". The protein's isotope effect, which reports on the shape of the energy well, is converted to H-bonding free energy by applying the scale factor. We estimate that amide-related H bonds (amide-carbonyl and amide-water) contribute favorably to protein stability by approximately 30-50 kcal/mol in lambda repressor, GCN4 coiled coil, and cytochrome c but unfavorably by approximately 6 kcal/mol in ubiquitin. The results indicate that H-bond strength varies from one protein to another and presumably at different sites within the same protein.
authors.affiliation: Department of Chemistry, New York University, 100 Washington Place, New York, New York 10003, USA.
authors.forename: Zhengshuang
authors.initials: Z
authors.lastname: Shi
authors.forename: Bryan A
authors.initials: BA
authors.lastname: Krantz
authors.forename: Neville
authors.initials: N
authors.lastname: Kallenbach
authors.forename: Tobin R
authors.initials: TR
authors.lastname: Sosnick
mesh_terms: D000577:Amides / Q000737:chemistry; D000595:Amino Acid Sequence; D010582:Bacteriophage lambda; D003574:Cytochrome c Group / Q000737:chemistry; D004268:DNA-Binding Proteins; D003903:Deuterium / Q000737:chemistry*; D005656:Fungal Proteins / Q000737:chemistry; D006860:Hydrogen Bonding; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011489:Protein Denaturation; D017510:Protein Folding; D011494:Protein Kinases / Q000737:chemistry; D012097:Repressor Proteins / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins; D013816:Thermodynamics; D025801:Ubiquitin / Q000737:chemistry; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
chemical_list: D000577:Amides; D003574:Cytochrome c Group; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D010446:Peptide Fragments; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D003903:Deuterium; D011494:Protein Kinases