Determination of residue-specific acid dissociation constants for peptides by band-selective homonuclear-decoupled (1)H NMR.
Acid dissociation constants of side-chain acidic groups of amino acid residues in peptides can be determined by 1H NMR, provided resonances can be resolved for carbon-bonded reporter protons located near the acidic group. We report here that the increased resolution of the band-selective homonuclear-decoupled (BASHD) TOCSY experiment greatly extends the range of application of the NMR method for determination of residue-specific, side-chain acid dissociation constants of peptides that contain multiple residues of the same amino acid. Chemical shift-pH titration curves are obtained from cross-peaks for reporter protons in BASHD-TOCSY spectra measured as a function of pH. The method is based on using sequence-dependent differences in the chemical shifts of resonances for the backbone CalphaH protons and the increased resolution in BASHD-TOCSY spectra from collapse of CalphaH multiplets to singlets in the F1 dimension to resolve resonances for the side-chain reporter protons. Application of the method is demonstrated by determination of residue-specific pKA values for each of the side-chain ammonium groups of the six lysine residues in the hexadecapeptide Ac-SRGKAKVKAKVKDQTK-NH2. Chemical shift-pH titration curves were obtained for the lysine side-chain CepsilonH2 reporter protons from their resolved CalphaH-CepsilonH2 TOCSY cross-peaks in BASHD-TOCSY spectra. Relative acidities of the six ammonium groups were also determined from the residue specific chemical shift-pH titration data by a pH-independent method, and calculation of fractional concentrations of protonation microspecies using the residue-specific pKAs is also described.
Wang
Jing
J
Department of Chemistry, University of California, Riverside, CA 92521, USA.
Rabenstein
Dallas L
DL
D000143:Acids / Q000032:analysis* / Q000737:chemistry*; D000595:Amino Acid Sequence; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D011522:Protons
D000143:Acids; D010455:Peptides; D011522:Protons; D008239:Lysine