Tautomeric states of the active‐site histidines of phosphorylated and unphosphorylated III^Glc, a signal‐transducing protein from escherichia coli, using two‐dimensional heteronuclear NMR techniques

Abstract

IIIG'C is an 18.1-kDa signal-transducing phosphocarrier protein of the phosphoeno1pyruvate:glycose phosphotransferase system from Escherichia coli. The 'H, "N, and I3C histidine ring NMR signals of both the phosphorylated and unphosphorylated forms of IIIGLC have been assigned using two-dimensional IH-"N and 'H-I3C heteronuclear multiple-quantum coherence (HMQC) experiments and a two-dimensional I3C-I3C-'H correlation spectroscopy via J,, coupling experiment. The data were acquired on uniformly 15N-labeled and uniformly '5N/'3C-labeled protein samples. The experiments rely on one-bond and two-bond J couplings that allowed for assignment of the signals without the need for the analysis of through-space (nuclear Overhauser effect spectroscopy) correlations. The "N and 13C chemical shifts were used t o determine that His-75 exists predominantly in the NE'-H tautomeric state in both the phosphorylated and unphosphorylated forms of IIIG'C, and that His-90 exists primarily in the N6'-H state in the unphosphorylated protein. Upon phosphorylation of the NE* nitrogen of His-90, the N*' nitrogen remains protonated, resulting in the formation of a charged phospho-His-90 moiety. The 'H, "N, and 13C signals of the phosphorylated and unphosphorylated proteins showed only minor shifts in the pH range from 6.0 to 9.0. These data indicate that the pK, values for both His-75 and His-90 in IIIG'' and His-75 in phospho-IIIG'C are less than 5.0, and that the pK, value for phospho-His-90 is greater than 10. The results are presented in relation to previously obtained structural data on IIIG", and implications for proposed mechanisms of phosphoryl transfer are discussed.

Measurement of histidine pKavalues and tautomer populations in invisible protein states

“…A promising alternative for studies of rare conformers is to monitor the chemical shifts of the 13 C γ and 13 C δ2 imidazole carbons. Theoretical and experimental studies of model compounds show that the 13 C γ and 13 C δ2 nuclei, but not 13 C e1 , are sensitive to the histidine conformational state, with shift differences as large as 12 and 8 ppm, respectively (7,52,56,57). Although the 13 C e1 chemical shift is insensitive to the imidazole conformational equilibrium, the one-bond 13 C e1 -1 H e1 scalar coupling ( 1 J C«H« ) shows a relatively large variation of ∼12 Hz between neutral and cationic states with little or no difference between neutral tautomers (58,59) (Fig.…”

“…Such interactions in proteins manifest most often through side chains of amino acids that can assume different protonation states and hence different charged forms that, for example, guide an enzyme reaction to completion (3)(4)(5). Histidine side chains play a particularly important role in the protein structure-function paradigm because they can exist in one of a pair of neutral tautomeric states or as a charged conformer, they serve as multiple hydrogen bond acceptors and donors, they are often localized to active sites of enzymes where they are integral to catalysis (5,6), and their properties can be modified through metal binding and phosphorylation (7,8). Moreover, the pK a of the histidine imidazole moiety is usually close to physiological pH allowing it to serve as either an acid or as a base, which nature has exploited for a variety of different protein functions (3,9).…”

“…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).…”

NMR structure of the mengovirus Leader protein zinc‐finger domain

“…A Zn atom was added to the CYANA library as a replacement for the H c of residue Cys10, and specific distance constraints were imposed for the Zn(II) coordination by residues Cys10, Cys19, Cys22, and His12. Small 2-and 3-bond J couplings (12 Hz, 2 Hz) [14] observed in a long-range 15 The superposition of the 20 lowest-energy conformers is shown in Fig. 4A.…”