The pH dependence of nitrogen-15 NMR shifts and coupling constants in aqueous imidazole and 1-methylimidazole. Comments on estimation of tautomeric equilibrium constants for aqueous histidine

Measurement of histidine pKavalues and tautomer populations in invisible protein states

“…The pK a values so obtained agree well with a previous study that used the 15 N HMBC approach (55) and virtually identical pK a s and extrema were obtained when the scalar coupling data were supplemented by measured 13 C, 15 N, and 1 H shifts for each histidine. Although there is some variation in the exact limiting values on a per-residue basis, the cationic and neutral extrema converge to 220.6 ± 1.0 Hz and 207.5 ± 1.5 Hz, respectively (Fig.…”

“…Thus, significant rearrangements of the histidine side chains must accompany the I-to-N transition, leading to burial of the exposed H40 and H47 side chains. Global structural changes have also been noted, largely from analysis of backbone 1 H, 15 N, and 13 C CPMG relaxation dispersion profiles that provide chemical shifts of the excited state (68)(69)(70). These, in turn, can be recast in terms of residue-specific fractional helicity values (79) and order parameters reflecting amplitudes of motion (80), with low values corresponding to disorder.…”

“…With the advent of multidimensional NMR spectroscopy and the development of 15 N-labeling methods many studies now rely on recording long-range 1 H- 15 N correlation maps that connect histidine 15 N e2 and 15 N δ1 chemical shifts with nonexchangeable ring protons. Analysis of such spectra, using canonical 15 N chemical shifts for the tautomeric and cationic states of the side chain, provides an estimate of the relative populations of each conformer, with pK a values fit from the pH dependence of the shifts (7,15,16).…”

“…The cationic form is of course dominant at low pH, whereas the « form is favored ∼4:1 over the δ form at high pH, according to observations of the free amino acid in solution (52,53). These conformations are usually indistinguishable in crystal structures, but can be readily identified by heteronuclear multiplebond correlation (HMBC)-type NMR experiments which measure the imidazole ring 15 N chemical shifts (7). Studies of the free amino acid, model compounds, and proteins have led to a canonical set of 15 N imidazole chemical shifts whereby the protonated and deprotonated nitrogen in either the δ or « form resonates at 167.5 or 249.5 ppm (7), respectively, whereas in the cationic form the resonance frequencies of the 15 N e2 and 15 N δ1 nuclei are at ∼174 and 178 ppm, respectively (54,55).…”

“…These conformations are usually indistinguishable in crystal structures, but can be readily identified by heteronuclear multiplebond correlation (HMBC)-type NMR experiments which measure the imidazole ring 15 N chemical shifts (7). Studies of the free amino acid, model compounds, and proteins have led to a canonical set of 15 N imidazole chemical shifts whereby the protonated and deprotonated nitrogen in either the δ or « form resonates at 167.5 or 249.5 ppm (7), respectively, whereas in the cationic form the resonance frequencies of the 15 N e2 and 15 N δ1 nuclei are at ∼174 and 178 ppm, respectively (54,55). These reference shifts provide a basis for interpreting measured 15 N e2 and 15 N δ1 resonances in terms of fractional populations of δ, «, and + (p δ , p « , p + ), and the HMBC experiment has therefore become the method of choice for studies of histidine side chains in proteins.…”