Tautomeric states of the active-site histidines of phosphorylated and unphosphorylated IIIGlc, a signal-transducing protein from Escherichia coli, using two-dimensional heteronuclear NMR techniques.

IIIGlc is an 18.1-kDa signal-transducing phosphocarrier protein of the phosphoenolpyruvate:glycose phosphotransferase system from Escherichia coli. The 1H, 15N, and 13C histidine ring NMR signals of both the phosphorylated and unphosphorylated forms of IIIGlc have been assigned using two-dimensional 1H-15N and 1H-13C heteronuclear multiple-quantum coherence (HMQC) experiments and a two-dimensional 13C-13C-1H correlation spectroscopy via JCC coupling experiment. The data were acquired on uniformly 15N-labeled and uniformly 15N/13C-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 15N and 13C chemical shifts were used to determine that His-75 exists predominantly in the N epsilon 2-H tautomeric state in both the phosphorylated and unphosphorylated forms of IIIGlc, and that His-90 exists primarily in the N delta 1-H state in the unphosphorylated protein. Upon phosphorylation of the N epsilon 2 nitrogen of His-90, the N delta 1 nitrogen remains protonated, resulting in the formation of a charged phospho-His-90 moiety. The 1H, 15N, 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 alpha values for both His-75 and His-90 in IIIGlc and His-75 in phospho-IIIGlc are less than 5.0, and that the pK alpha value for phospho-His-90 is greater than 10. The results are presented in relation to previously obtained structural data on IIIGlc, and implications for proposed mechanisms of phosphoryl transfer are discussed.     

Chain-selective isotopic labeling for NMR studies of large multimeric proteins: application to hemoglobin

Multidimensional, multinuclear NMR has the potential to elucidate the mechanisms of allostery and cooperativity in multimeric proteins under near-physiological conditions. However, NMR studies of proteins made up of non-equivalent subunits face the problem of severe resonance overlap, which can prevent the unambiguous assignment of resonances, a necessary step in interpreting the spectra. We report the application of a chain-selective labeling technique, in which one type of subunit is labeled at a time, to carbonmonoxy-hemoglobin A (HbCO A). This labeling method can be used to extend previous resonance assignments of key amino acid residues, which are important to the physiological function of hemoglobin. Among these amino acid residues are the surface histidyls, which account for the majority of the Bohr effect. In the present work, we report the results of two-dimensional heteronuclear multiple quantum coherence (HMQC) experiments performed on recombinant (15)N-labeled HbCO A. In addition to the C2-proton (H epsilon(1)) chemical shifts, these spectra also reveal the corresponding C4-proton (H delta(2)) resonances, correlated with the N epsilon(2) and N delta(1) chemical shifts of all 13 surface histidines per alpha beta dimer. The HMQC spectrum also allows the assignment of the H delta(1), H epsilon(1), and N epsilon(1) resonances of all three tryptophan residues per alpha beta dimer in HbCO A. These results indicate that heteronuclear NMR, used with chain-selective isotopic labeling, can provide resonance assignments of key regions in large, multimeric proteins, suggesting an approach to elucidating the solution structure of hemoglobin, a protein with molecular weight 64.5 kDa.     

1H-NMR study on the tautomerism of the imidazole ring of histidine residues. II. Microenvironments of histidine-12 and histidine-119 of bovine pancreatic ribonuclease A

The NMR titration curves of proton chemical shifts were observed for the C2 protons of histidine residues in intact bovine pancreatic RNAase A (EC 3.1.27.5) and carboxyalkylated RNAase A. By comparing the methyl region of NMR spectra, the 250-340 nm region of circular dichoic spectra, and the NMR titration curves of tyrosine ring protons among intact and modified RNAase A, it was ascertained that the carboxyalkylation of histidine residues at position 12 or 119 did not make any appreciable conformational changes to RNAase A. With the pK values determined for intact and modified RNAase A, the microscopic pK values and molar ratios of tautomers were estimated for His-12 and His-119 by means of the procedure described in the preceding paper. The estimated microscopic pK values of tautomers were 6.2 for the N1-H tautomer of His-12, more than 8 for the N3-H tautomer of His-12, 7.0 for the N1-H tautomer of His-119, and 6.4 for the N3-H tautomer of His-119, respectively. These values were interpreted in terms of the microscopic environments surrounding the histidine residues. The microscopic structure estimated in the present study was discussed, comparing it with those from X-ray crystallography and hydrogen-tritium (or hydrogen-deuterium) exchange technique.     

The roles of Glu-327 and His-446 in the bisphosphatase reaction of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase probed by NMR spectroscopic and mutational analyses of the enzyme in the transient phosphohistidine intermediate complex

The bisphosphatase domain derived from the rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was studied by 1H-13C HMQC NMR spectroscopy of the histidine C2' and H2' nuclei. The bacterially expressed protein was specifically labeled with 13C at the ring C2' position of the histidines. Each of the seven histidine residues gave rise to a single cross-peak in the HMQC spectra, and these were assigned by use of a series of histidine-to-alanine point mutants. His-304, His-344, and His-469 exhibit 13C and 1H resonances that titrated with pH, while the remaining histidine-associated resonances did not. The 13C and 1H chemical shifts indicate that at neutral pH, His-304 and His-446 are deprotonated, while His-469 is protonated. The pKa of His-344 was determined to be 7.04. The 13C chemical shifts suggest that the deprotonated His-258 exists as the N1' tautomer, while His-392 and His-419 are protonated in the resting, wild-type enzyme. Mutation of the remaining member of the catalytic triad, Glu-327, to alanine in the resting enzyme caused an upfield shift of 1.58 and 1.30 ppm in the 1H and 13C dimensions, respectively, and significant narrowing of the His-258 cross-peak. Mutation of His-446 to alanine produced perturbations of the His-258 cross-peak that were similar to those detected in the E327A mutant. The His-392 resonances were also shifted by the E327A and H446A mutations. These observations strongly suggest that residues His-258, Glu-327, His-392, and His-446 exist within a network of interacting residues that encompasses the catalytic site of the bisphosphatase and includes specific contacts with the C-terminal regulatory region of the enzyme. The specifically 13C-labeled bisphosphatase was monitored during turnover by HMQC spectra acquired from the transient N3' phosphohistidine intermediate complex in the wild-type enzyme, the E327A mutant, and the H446A mutant. These complexes were formed during reaction with the physiological substrate fructose-2, 6-bisphosphate. Upon formation of the phosphohistidine at His-258, the 13C and 1H resonances of this residue were shifted downfield by 1.7 and 0.31 ppm, respectively, in the wild-type enzyme. The upfield shifts of the His-258 resonances in the E327A and H446A mutant resting enzymes were reversed when the phosphohistidine was formed, generating spectra very similar to that of the wild-type enzyme in the intermediate complex. In contrast, the binding of fructose-6-phosphate, the reaction product, to the resting enzyme did not promote significant changes in the histidine-associated resonances in either the wild-type or the mutant enzymes. The interpretation of these data within the context of the X-ray crystal structures of the enzyme is used to define the role of Glu-327 in the catalytic mechanism of the bisphosphatase and to identify His-446 as a putative link in the chain of molecular events that results in activation of the bisphosphatase site by cAMP-dependent phosphorylation of the hepatic bifunctional enzyme.     

Complete sequential 1H and 15N nuclear magnetic resonance assignments and solution secondary structure of the blue copper protein azurin from Pseudomonas aeruginosa.

Complete sequential 1H and 15N resonance assignments for the reduced Cu(I) form of the blue copper protein azurin (M(r) 14,000, 128 residues) from Pseudomonas aeruginosa have been obtained at pH 5.5 and 40 degrees C by using homo- and heteronuclear two-dimensional (2D) and three-dimensional (3D) nuclear magnetic resonance spectroscopic experiments. Combined analysis of a 3D homonuclear 1H Hartmann-Hahn nuclear Overhauser (3D 1H HOHAHA-NOESY) spectrum and a 3D heteronuclear 1H nuclear Overhauser 1H[15N] single-quantum coherence (3D 1H[15N] NOESY-HSQC) spectrum proved especially useful. The latter spectrum was recorded without irradiation of the water signal and provided for differential main chain amide (NH) exchange rates. NMR data were used to determine the secondary structure of azurin in solution. Comparison with the secondary structure of azurin obtained from X-ray analysis shows a virtually complete resemblance; the two beta-sheets and a 3(10)-alpha-3(10) helix are preserved at 40 degrees C, and most loops contain well-defined turns. Special findings are the unexpectedly slow exchange of the Asn-47 and Phe-114 NH's and the observation of His-46 and His-117 N epsilon 2H resonances. The implications of these observations for the assignment of azurin resonance Raman spectra, the rigidity of the blue copper site, and the electron transfer mechanism of azurin are discussed.     

Structural comparison of phosphorylated and unphosphorylated forms of IIIGlc, a signal-transducing protein from Escherichia coli, using three-dimensional NMR techniques.

The 18.1-kDa protein IIIGlc from Escherichia coli acts as both a phosphocarrier protein in the phosphoenolpyruvate:glycose phosphotransferase system (PTS) and as a signal-transducing protein with respect to the uptake of non-PTS sugars. Phosphorylation of IIIGlc at the N epsilon (N3) position of His-90 was effected through a regeneration system that included MgCl2, DTT, excess PEP, and catalytic amounts of Enzyme I and HPr. NH, 15N, and 13C alpha signal assignments for P-IIIGlc were made through comparison of 15N-1H correlation spectra (HSQC) of uniformly 15N-labeled preparations of phosphorylated and unphosphorylated protein and through analysis of three-dimensional triple-resonance HNCA spectra of P-IIIGlc uniformly labeled with both 15N and 13C. Backbone and side-chain 1H and 13C beta signals were assigned using 3D heteronuclear HCCH-COSY and HCCH-TOCSY spectra of P-IIIGlc. Using this approach, the assignments were made without reference to nuclear Overhauser effect data or assumptions regarding protein structure. The majority of NH, 15N, H alpha, and 13C alpha chemical shifts measured for P-IIIGlc were identical to those obtained for the unphosphorylated protein [Pelton, J. G., Torchia, D. A., Meadow, N. D., Wong, C.-Y., & Roseman, S. (1991) Biochemistry 30, 10043]. Those signals that exhibited shifts corresponded to residues within four segments (1) Leu-87-Gly-100, (2) Val-36-Val-46, (3) His-75-Ser-78, and (4) Ala-131-Val-138. These four segments are in close proximity to the active site residues His-75 and His-90 in the unphosphorylated protein [Worthylake, D., Meadow, N. D., Roseman, S., Liao, D., Hertzberg, O., & Remington, S.J. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 10382], and the chemical shift data provide strong evidence that if any structural changes accompany phosphorylation, they are confined to residues in these four segments. This conclusion is confirmed by comparing NOEs observed in 3D 15N/13C NOESY-HMQC spectra of the two forms of the protein. No NOE differences are seen for residues having the same chemical shifts in IIIGlc and P-IIIGlc. Furthermore, with the exception of residues Ala-76, Asp-94, and Val-96, the NOEs of residues (in the four segments) which exhibited chemical shift differences also had the same NOEs in IIIGlc and P-IIIGlc. In the case of residues Ala-76, Asp-94, and Val-96, minor differences in NOEs, corresponding to interproton distances changes of less than 1.5 A, were observed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Assignment of the side-chain 1H and 13C resonances of interleukin-1 beta using double- and triple-resonance heteronuclear three-dimensional NMR spectroscopy

The assignment of the aliphatic 1H and 13C resonances of IL-1 beta, a protein of 153 residues and molecular mass 17.4 kDa, is presented by use of a number of novel three-dimensional (3D) heteronuclear NMR experiments which rely on large heteronuclear one-bond J couplings to transfer magnetization and establish through-bond connectivities. These 3D NMR experiments circumvent problems traditionally associated with the application of conventional 2D 1H-1H correlation experiments to proteins of this size, in particular the extensive chemical shift overlap which precludes the interpretation of the spectra and the reduced sensitivity arising from 1H line widths that are often significantly larger than the 1H-1H J couplings. The assignment proceeds in two stages. In the first step the 13C alpha chemical shifts are correlated with the NH and 15N chemical shifts by a 3D triple-resonance NH-15N-13C alpha (HNCA) correlation experiment which reveals both intraresidue NH(i)-15N(i)-13C alpha (i) and some weaker interresidue NH(i)-15N(i)-C alpha (i-1) correlations, the former via intraresidue one-bond 1JNC alpha and the latter via interresidue two-bond 2JNC alpha couplings. As the NH, 15N, and C alpha H chemical shifts had previously been sequentially assigned by 3D 1H Hartmann-Hahn 15N-1H multiple quantum coherence (3D HOHAHA-HMQC) and 3D heteronuclear 1H nuclear Overhauser 15N-1H multiple quantum coherence (3D NOESY-HMQC) spectroscopy [Driscoll, P.C., Clore, G.M., Marion, D., Wingfield, P.T., & Gronenborn, A.M. (1990) Biochemistry 29, 3542-3556], the 3D triple-resonance HNCA correlation experiment permits the sequence-specific assignments of 13C alpha chemical shifts in a straightforward manner. The second step involves the identification of side-chain spin systems by 3D 1H-13C-13C-1H correlated (HCCH-COSY) and 3D 1H-13C-13C-1H total correlated (HCCH-TOCSY) spectroscopy, the latter making use of isotropic mixing of 13C magnetization to obtain relayed connectivities along the side chains. Extensive cross-checks are provided in the assignment procedure by examination of the connectivities between 1H resonances at all the corresponding 13C shifts of the directly bonded 13C nuclei. In this manner, we were able to obtain complete 1H and 13C side-chain assignments for all residues, with the exception of 4 (out of a total of 15) lysine residues for which partial assignments were obtained. The 3D heteronuclear correlation experiments described are highly sensitive, and the required set of three 3D spectra was recorded in only 1 week of measurement time on a single uniformly 15N/13C-labeled 1.7 mM sample of interleukin-1 beta.(ABSTRACT TRUNCATED AT 400 WORDS)     

Tautomerism,acid-base equilibria,and H-bonding of the six histidines in subtilisin BPN' by NMR

We have determined by (15)N, (1)H, and (13)C NMR, the chemical behavior of the six histidines in subtilisin BPN' and their PMSF and peptide boronic acid complexes in aqueous solution as a function of pH in the range of from 5 to 11, and have assigned every (15)N, (1)H, C(epsilon 1), and C(delta2) resonance of all His side chains in resting enzyme. Four of the six histidine residues (17, 39, 67, and 226) are neutrally charged and do not titrate. One histidine (238), located on the protein surface, titrates with pK(a) = 7.30 +/- 0.03 at 25 degrees C, having rapid proton exchange, but restricted mobility. The active site histidine (64) in mutant N155A titrates with a pK(a) value of 7.9 +/- 0.3 and sluggish proton exchange behavior, as shown by two-site exchange computer lineshape simulation. His 64 in resting enzyme contains an extremely high C(epsilon 1)-H proton chemical shift of 9.30 parts per million (ppm) owing to a conserved C(epsilon 1)-H(.)O=C H-bond from the active site imidazole to a backbone carbonyl group, which is found in all known serine proteases representing all four superfamilies. Only His 226, and His 64 at high pH, exist as the rare N(delta1)-H tautomer, exhibiting (13)C(delta1) chemical shifts approximately 9 ppm higher than those for N(epsilon 2)-H tautomers. His 64 in the PMSF complex, unlike that in the resting enzyme, is highly mobile in its low pH form, as shown by (15)N-(1)H NOE effects, and titrates with rapid proton exchange kinetics linked to a pK(a) value of 7.47 +/- 0.02.     

NMR study of the positions of His-12 and His-119 in the ribonuclease A-uridine vanadate complex.

The binding of uridine vanadate to ribonuclease A has been investigated by one- and two-dimensional 1H NMR. The homonuclear Nuclear Overhauser and exchange spectroscopy spectrum of the uridine vanadate/RNase A complex exhibits cross peaks between both the C5H and C6H protons of uridine vanadate and the H epsilon 1 proton of His-12 of ribonuclease A. These cross peaks suggest that the H epsilon 1 proton of His-12 is in the vicinity of the uracil base of uridine vanadate, as observed in the crystallographic structure of the uridine vanadate/RNase A complex. However, no cross peaks are observed between the C5H and C6H protons of uridine vanadate and the H epsilon 1 proton of His-119 of ribonuclease A, although they were predicted based upon the distances calculated from coordinates of the crystallographic structure of the complex. These results suggest that there is a significant difference between the positioning of the His-119 side chain in the solution and in the crystallographic structures.     

NMR studies of the exocyclic 1,N6-ethenodeoxyadenosine adduct (epsilon dA) opposite deoxyguanosine in a DNA duplex. Epsilon dA(syn).dG(anti) pairing at the lesion site

Proton NMR studies are reported on the complementary d(C-A-T-G-G-G-T-A-C).d(G-T-A-C-epsilon A-C-A-T-G) nonanucleotide duplex (designated epsilon dA.dG 9-mer duplex), which contains exocyclic adduct 1,N6-ethenodeoxyadenosine positioned opposite deoxyguanosine in the center of the helix. The present study focuses on the alignment of dG5 and epsilon dA14 at the lesion site in the epsilon dA.dG 9-mer duplex at neutral pH. This alignment has been characterized by monitoring the NOEs originating from the NH1 proton of dG5 and the H2, H5, and H7/H8 protons of epsilon dA14 in the central d(G4-G5-G6).d(C13-epsilon A14-C15) trinucleotide segment of the epsilon dA.dG 9-mer duplex. These NOE patterns establish that epsilon dA14 adopts a syn glycosidic torsion angle that positions the exocyclic ring toward the major groove edge while all the other bases including dG5 adopt anti glycosidic torsion angles. We detect a set of intra- and interstrand NOEs between protons (exchangeable and nonexchangeable) on adjacent residues in the d(G4-G5-G6).d(C13-epsilon A14-C15) trinucleotide segment which establish formation of right-handed helical conformations on both strands and stacking of the dG5(anti).epsilon dA14(syn) pair between stable dG4.dC15 and dG6.dC13 pairs. The energy-minimized conformation of the central d(G4-G5-G6).d(C13-epsilon A14-C15) segment establishes that the dG5(anti).epsilon dA14(syn) alignment is stabilized by two hydrogen bonds from the NH1 and NH2-2 of dG5(anti) to N9 and N1 of epsilon dA14(syn), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Three-dimensional 15N-1H-1H and 15N-13C-1H nuclear-magnetic resonance studies of HPr a central component of the phosphoenolpyruvate-dependent phosphotransferase system from Escherichia coli. Assignment of backbone resonances.

We have performed three-dimensional NMR studies on a central component of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli, denoted as HPr. The protein was uniformly enriched with 15N and 13C to overcome spectral overlap. Complete assignments were obtained for the backbone 1H, 15N and 13C resonances, using three-dimensional heteronuclear 1H NOE 1H-15N multiple-quantum coherence spectroscopy (3D-NOESY-HMQC) and three-dimensional heteronuclear total correlation 1H-15N multiple-quantum coherence spectroscopy (3D-TOCSY-HMQC) experiments on 15N-enriched HPr and an additional three-dimensional triple-resonance 1HN-15N-13C alpha correlation spectroscopy (HNCA) experiment on 13C, 15N-enriched HPr. Many of the sequential backbone 1H assignments, as derived from two-dimensional NMR studies [Klevit, R.E., Drobny, G.P. & Waygood, E.B. (1986) Biochemistry 25, 7760-7769], were corrected. Almost all discrepancies are in the helical regions, leaving the published antiparallel beta-sheet topology almost completely intact.     

Determination of the three-dimensional solution structure of the histidine-containing phosphocarrier protein HPr from Escherichia coli using multidimensional NMR spectroscopy.

We recorded several types of heteronuclear three-dimensional (3D) NMR spectra on 15N-enriched and 13C/15N-enriched histidine-containing phosphocarrier protein, HPr, to extend the backbone assignments [van Nuland, N. A. J., van Dijk, A. A., Dijkstra, K., van Hoesel, F. H. J., Scheek, R. M. & Robillard, G. T. (1992) Eur. J. Biochem, 203, 483-491] to the side-chain 1H,15N and 13C resonances. From both 3D heteronuclear 1H-NOE 1H-13C and 1H-NOE 1H-15N multiple-quantum coherence (3D-NOESY-HMQC) and two-dimensional (2D) homonuclear NOE spectra, more than 1200 NOE were identified and used in a step-wise structure refinement process using distance geometry and restrained molecular dynamics involving a number of new features. A cluster of nine structures, each satisfying the set of NOE restraints, resulted from this procedure. The average root-mean-square positional difference for the C alpha atoms is less than 0.12 nm. The secondary structure topology of the molecule is that of an open-face beta sandwich formed by four antiparallel beta strands packed against three alpha helices, resembling the recently published structure of Bacillus subtilis HPr, determined by X-ray crystallography [Herzberg, O., Reddy, P., Sutrina, S., Saier, M. H., Reizer, J. & Kapafia, G. (1992) Proc. Natl, Acad. Sci. USA 89, 2499-2503).     

NMR assignments of the four histidines of staphylococcal nuclease in native and denatured states

NMR signals from all four histidine ring C epsilon protons and three of the four histidine C delta protons in the protein staphylococcal nuclease have been assigned by comparing spectra of the wild-type (Foggi strain) protein to spectra of three variants that each lack a different histidine residue. All proteins studied were cloned and overproduced in Escherichia coli. The NMR spectra of the three mutant proteins (H8R, H46Y, and H124L) used to make these assignments were similar to one another and to those of the wild type, except for signals from the mutated residues. The pKa values of those histidines conserved between the wild type and the mutants remained essentially unchanged. Multiple histidine C epsilon proton resonances due to non-native forms of nuclease were observed in both thermally induced and acid-induced unfolding. Residue-specific assignments of H epsilon protons in the thermally denatured forms of the mutant H46Y were obtained from connectivities to the native state by saturation transfer.     

Assignment of the aliphatic 1H and 13C resonances of the Bacillus subtilis glucose permease IIA domain using double- and triple-resonance heteronuclear three-dimensional NMR spectroscopy.

Nearly complete assignment of the aliphatic 1H and 13C resonances of the IIAglc domain of Bacillus subtilis has been achieved using a combination of double- and triple-resonance three-dimensional (3D) NMR experiments. A constant-time 3D triple-resonance HCA(CO)N experiment, which correlates the 1H alpha and 13C alpha chemical shifts of one residue with the amide 15N chemical shift of the following residue, was used to obtain sequence-specific assignments of the 13C alpha resonances. The 1H alpha and amide 15N chemical shifts had been sequentially assigned previously using principally 3D 1H-15N NOESY-HMQC and TOCSY-HMQC experiments [Fairbrother, W. J., Cavanagh, J., Dyson, H. J., Palmer, A. G., III, Sutrina, S. L., Reizer, J., Saier, M. H., Jr., & Wright, P. E. (1991) Biochemistry 30, 6896-6907]. The side-chain spin systems were identified using 3D HCCH-COSY and HCCH-TOCSY spectra and were assigned sequentially on the basis of their 1H alpha and 13C alpha chemical shifts. The 3D HCCH and HCA(CO)N experiments rely on large heteronuclear one-bond J couplings for coherence transfers and therefore offer a considerable advantage over conventional 1H-1H correlation experiments that rely on 1H-1H 3J couplings, which, for proteins the size of IIAglc (17.4 kDa), may be significantly smaller than the 1H line widths. The assignments reported herein are essential for the determination of the high-resolution solution structure of the IIAglc domain of B. subtilis using 3D and 4D heteronuclear edited NOESY experiments; these assignments have been used to analyze 3D 1H-15N NOESY-HMQC and 1H-13C NOESY-HSQC spectra and calculate a low-resolution structure [Fairbrother, W. J., Gippert, G. P., Reizer, J., Saier, M. H., Jr., & Wright, P. E. (1992) FEBS Lett. 296, 148-152].     

The tautomeric state of histidines in myoglobin

1H-15N HMQC spectra were collected on 15N-labeled sperm whale myoglobin (Mb) to determine the tautomeric state of its histidines in the neutral form. By analyzing metaquoMb and metcyanoMb data sets collected at various pH values, cross-peaks were assigned to the imidazole rings and their patterns interpreted. Of the nine histidines not interacting with the heme in sperm whale myoglobin, it was found that seven (His-12, His-48, His-81, His-82, His-113, His-116, and His-119) are predominantly in the N epsilon2H form with varying degrees of contribution from the Ndelta1 H form. The eighth, His-24, is in the Ndelta1H state as expected from the solid state structure. 13C correlation spectra were collected to probe the state of the ninth residue (His-36). Tentative interpretation of the data through comparison with horse Mb suggested that this ring is predominantly in the Ndelta1H state. In addition, signals were observed from the histidines associated with the heme (His-64, His-93, and His-97) in the 1H-15N HMQC spectra of the metcyano form. In several cases, the tautomeric state of the imidazole ring could not be derived from inspection of the solid state structure. It was noted that hydrogen bonding of the ring was not unambiguously reflected in the nitrogen chemical shift. With the experimentally determined tautomeric state composition in solution, it will be possible to broaden the scope of other studies focused on the electrostatic contribution of histidines to the thermodynamic properties of myoglobin.     

Disruption of an intermonomer salt bridge in the p53 tetramerization domain results in an increased propensity to form amyloid fibrils

We describe in molecular detail how disruption of an intermonomer salt bridge (Arg337-Asp352) leads to partial destabilization of the p53 tetramerization domain and a dramatically increased propensity to form amyloid fibrils. At pH 4.0 and 37 degrees C, a p53 tetramerization domain mutant (p53tet-R337H), associated with adrenocortical carcinoma in children, readily formed amyloid fibrils, while the wild-type (p53tet-wt) did not. We characterized these proteins by equilibrium denaturation, 13C(alpha) secondary chemical shifts, (1H)-15N heteronuclear NOEs, and H/D exchange. Although p53tet-R337H was thermodynamically less stable, NMR data indicated that the two proteins had similar secondary structure and molecular dynamics. NMR derived pK(a) values indicated that at low pH the R337H mutation partially disrupted an intermonomer salt bridge. Backbone H/D exchange results showed that for at least a small population of p53tet-R337H molecules disruption of this salt bridge resulted in partial destabilization of the protein. It is proposed that this decrease in p53tet-R337H stability resulted in an increased propensity to form amyloid fibrils.     

Complete resonance assignment for the polypeptide backbone of interleukin 1 beta using three-dimensional heteronuclear NMR spectroscopy

The complete sequence-specific assignment of the 15N and 1H backbone resonances of the NMR spectrum of recombinant human interleukin 1 beta (153 residues, Mr = 17,400) has been obtained by using primarily 15N-1H heteronuclear three-dimensional (3D) NMR techniques in combination with 15N-1H heteronuclear and 1H homonuclear two-dimensional NMR. The fingerprint region of the spectrum was analyzed by using a combination of 3D heteronuclear 1H Hartmann-Hahn 15N-1H multiple quantum coherence (3D HOHAHA-HMQC) and 3D heteronuclear 1H nuclear Overhauser 15N-1H multiple quantum coherence (3D NOESY-HMQC) spectroscopies. We show that the problems of amide NH and C alpha H chemical shift degeneracy that are prevalent for proteins of this size are readily overcome by using the 3D heteronuclear NMR technique. A doubling of some peaks in the spectrum was found to be due to N-terminal heterogeneity of the 15N-labeled protein, corresponding to a mixture of wild-type and des-Ala-1-interleukin 1 beta. The complete list of 15N and 1H assignments is given for all the amide NH and C alpha H resonances of all non-proline residues, as well as the 1H assignments for some of the amino acid side chains. This first example of the sequence-specific assignment of a protein using heteronuclear 3D NMR provides a basis for further conformational and dynamic studies of interleukin 1 beta.     

Structure analyses of DNA oligomers by use of heteronuclear 2D NMR.

DNA oligomer d(CGGAAGACTCTCCTCCG):d(CGGAGGAGAGTCTTCCG) named UASG (17mer M.W. = 11 kDa) was studied by 1H NMR and heteronuclear two dimensional (2D) NMR. All the labile protons and half of the non-exchangeable protons were assigned by use of conventional 1H 2D experiments including NOESY using 1-1 echo excitation for water suppression. Signal degeneracy in the sugar proton region made it difficult to make assignments of the remaining half of the non-exchangeable protons of the oligomer in 1H 2D spectra. Here we report a new strategy using 1H/13C and 1H/31P heteronuclear single-quantum correlation spectroscopy combined with homonuclear three dimensional NOESY-TOCSY. By this strategy, most of the proton resonances of the oligomer have been assigned, and it turned out that the whole conformation of the oligomer is B-form like.     

Mechanism of the bisphosphatase reaction of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase probed by (1)H-(15)N NMR spectroscopy

The histidines in the bisphosphatase domain of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase were labeled with (15)N, both specifically at N1' and globally, for use in heteronuclear single quantum correlation (HSQC) NMR spectroscopic analyses. The histidine-associated (15)N resonances were assigned by correlation to the C2' protons which had been assigned previously [Okar et al., Biochemistry 38, 1999, 4471-79]. Acquisition of the (1)H-(15)N HSQC from a phosphate-free sample demonstrated that the existence of His-258 in the rare N1' tautomeric state is dependent upon occupation of the phosphate binding site filled by the O2 phosphate of the substrate, fructose-2,6-bisphosphate, and subsequently, the phosphohistidine intermediate. The phosphohistidine intermediate is characterized by two hydrogen bonds involving the catalytic histidines, His-258 and His-392, which are directly observed at the N1' positions of the imidazole rings. The N1' of phospho-His-258 is protonated ((1)H chemical shift, 14.0 ppm) and hydrogen bonded to the backbone carbonyl of Gly-259. The N1' of cationic His-392 is hydrogen bonded ((1)H chemical shift, 13.5 ppm) to the phosphoryl moiety of the phosphohistidine. The existence of a protonated phospho-His-258 intermediate and the observation of a fairly strong hydrogen bond to the same phosphohistidine implies that hydrolysis of the covalent intermediate proceeds without any requirement for an "activated" water. Using the labeled histidines as probes of the catalytic site mutation of Glu-327 to alanine revealed that, in addition to its function as the proton donor to fructose-6-phosphate during formation of the transient phosphohistidine intermediate at the N3' of His-258, this residue has a significant role in maintaining the structural integrity of the catalytic site. The (1)H-(15)N HSQC data also provide clear evidence that despite being a surface residue, His-446 has a very acidic pK(a), much less than 6.0. On the basis of these observations a revised mechanism for fructose-2,6-bisphosphatase that is consistent with all of the previously published kinetic data and X-ray crystal structures is proposed. The revised mechanism accounts for the structural and kinetic consequences produced by mutation of the catalytic histidines and Glu-327. It also provides the basis for a hypothetical mechanism of bisphosphatase activation by cAMP-dependent phosphorylation of Ser-32, which is located in the N-terminal kinase domain.