title: Measurement of side-chain carboxyl pK(a) values of glutamate and aspartate residues in an unfolded protein by multinuclear NMR spectroscopy.
abstract: A triple-resonance NMR pulse scheme is presented for measuring aspartic and glutamic acid side-chain pK(a) values in unfolded protein states where chemical shift overlap is limiting. The experiment correlates side-chain carboxyl carbon chemical shifts of these residues with the backbone amide proton chemical shift of the following residue. The methodology is applied to an (15)N, (13)C labeled sample of the N-terminal SH3 domain of the Drosophila protein drk, which exists in equilibrium between folded (F(exch)) and unfolded (U(exch)) states under nondenaturing conditions. Residue-specific pK(a) values of side-chain carboxyl groups are presented for the first time for an unfolded protein (drk U(exch) state), determined from a pH titration. Results indicate that deviations from pK(a) values measured for model compounds are likely due to local effects, while long-range electrostatic interactions appear to be of minor importance for this protein.
authors: Structural Biology and Biochemistry Program, Hospital for Sick Children, Toronto, Ontario, Canada.
authors: Martin
authors: M
authors: Tollinger
authors: Julie D
authors: JD
authors: Forman-Kay
authors: Lewis E
authors: LE
authors: Kay
mesh_terms: D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry*; D002247:Carbon Isotopes; D004330:Drosophila; D029721:Drosophila Proteins; D005583:Fourier Analysis; D018698:Glutamic Acid / Q000737:chemistry*; D019476:Insect Proteins / Q000737:chemistry*; D007700:Kinetics; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D017510:Protein Folding; D018909:src Homology Domains
chemical_list: D002247:Carbon Isotopes; D029721:Drosophila Proteins; D019476:Insect Proteins; D009587:Nitrogen Isotopes; C080530:drk protein, Drosophila; D001224:Aspartic Acid; D018698:Glutamic Acid