NMR study of the phosphate-binding loops of Thermus thermophilus elongation factor Tu.

The phosphoryl-binding loops in the guanosine diphosphate binding domain of elongation factor Tu were studied by 15N heteronuclear proton-observe NMR methods. Five proton resonances were found below 10.5 ppm. One of these was assigned to the amide group of Lys 24, which is a conserved residue in the phosphoryl-binding concensus loop of purine nucleotide binding proteins. The uncharacteristic downfield proton shift is attributed to a strong hydrogen bond with a phosphate oxygen. The amide protons from the homologous lysines in N-ras p21 [Redfield, A.G., & Papastavros, M.Z. (1990) Biochemistry 29, 3509-3514] and the catalytic domain of Escherichia coli elongation factor Tu [Lowry, D.F., Cool, R.H., Redfield, A.G., & Parmeggiani, A. (1991) Biochemistry 30, 10872-10877] also resonate downfield in similar positions. We propose that the downfield shift of this lysine amide proton is a spectral marker for this class of proteins. We also have studied the temperature dependence of the downfield resonances and find a possible conformation change at 40 degrees C.     

Nuclear magnetic resonance study of exchangeable protons in ferrocytochrome c

Five well-resolved exchangeable proton resonances have been observed in horse ferrocytochrome c in the low-field region between ?10.0 and ?12.0 ppm. A resonance at ?11.6 ppm is assigned to an amino proton of His 26 on the basis of its magnetic field position, pH dependence and its correlation with histidine at this position in eight species. A resonance at ?10.9 ppm observed in horse and donkey ferrocytochrome c is assigned to a hydrogen-bonded ?-amino proton of Lys 60. This resonance shifts upfield with increasing salt and decreasing pH, with the shifts increasing as one goes from chloride to bromide to iodide. With less assurance, a resonance at ?10.6 ppm is assigned to the indole amino of Trp 59 and a resonance at ?10.3 ppm to the amino proton of the His 18. These resonances have been used to study the binding of small anions to ferrocytochrome c, the results of which show that inorganic phosphate, ADP and ATP all bind in the immediate vicinity of His 26, and that the inorganic phosphate has the greatest effect upon the imidazole amino exchange time.     

A proton nuclear Overhauser effect investigation of the subunit interfaces in human normal adult hemoglobin

High-resolution proton nuclear magnetic resonance spectroscopy and nuclear Overhauser effects for the low-field exchangeable proton resonances of human normal adult hemoglobin in aqueous solvents are being used to confirm and extend the assignments of these resonances to specific protons at the intersubunit interfaces of the molecule. Most of these exchangeable proton resonances of human normal adult hemoglobin have been found to be absent in the spectra of isolated alpha or beta subunits. This finding indicates that they are specific spectral markers for the quaternary structure of the hemoglobin tetramer. Based on the nuclear Overhauser effect results, we have assigned the exchangeable proton resonance at +7.4 ppm downfield from H2O to the hydrogen-bonded proton between alpha 103(G10)His and beta 108(G10)Asn at the alpha 1 beta 1 interface. The nuclear Overhauser effect results have also confirmed the assignments of the exchangeable proton resonances at +9.4 and +8.2 ppm downfield from H2O previously proposed by workers in this laboratory based on a comparison of human normal adult hemoglobin and appropriate mutant hemoglobins. This independent confirmation of previously proposed assignments is necessary in view of the possible long-range conformational effects of single amino-acid substitutions in mutant hemoglobin molecules.     

Proton magnetic resonance spectra of adrenodoxin: features of the aromatic region

This paper presents the first 1H-NMR spectra of the aromatic region of adrenodoxin, a mammalian mitochondrial 2Fe-2S non-heme iron ferredoxin. One-dimensional proton NMR spectra of both reduced and oxidized adrenodoxin were recorded as a function of pH. Resonances due to two of the three histidines of adrenodoxin gave sharp signals in the one-dimensional proton NMR spectra. The pKa values of the resolved histidine resonances in the oxidized protein were 6.64 +/- 0.03 and 6.12 +/- 0.06. These values were unchanged when adrenodoxin was reduced by the addition of sodium dithionite. In addition, the oxidized protein showed a broadened histidine C-2H resonance with a pKa value of approx. 7. This resonance was not apparent in the spectra of the reduced protein. The resonances due to the single tyrosine in adrenodoxin were identified using convolution difference spectroscopy. In addition, a two-dimensional Fourier-transform double quantum filtered (proton, proton) chemical shift correlated (DQF-COSY) spectrum of oxidized adrenodoxin was obtained. The cross peaks of the resonances due to the tyrosine, the four phenylalanines, and two of the three histidines of adrenodoxin were resolved in the DQF-COSY spectrum. Reduction of the protein caused several changes in the aromatic region of the NMR spectra. The resonances assigned to the C2 proton of the histidine with a pKa of 6.6 shifted upfield approx. 0.15 ppm. In addition, when the protein was reduced one of the resonances assigned to a phenylalanine residue with a chemical shift of 7.50 ppm appeared to move downfield to 7.82 ppm.(ABSTRACT TRUNCATED AT 250 WORDS)     

A proton nuclear magnetic resonance investigation of proximal histidyl residues in human normal and abnormal hemoglobins. A probe for the heme pocket.

Proton nuclear magnetic resonance spectroscopy at 250 MHz has been used to investigate the conformations of proximal histidyl residues of human normal adult hemoglobin, hemoglobin Kempsey [beta 99(G1) Asp leads to Asn], hemoglobin Osler [beta 145(HC2) Tyr leads to Asp], and hemoglobin McKees Rocks [beta 145(HC2) Tyr leads to Term] around neutral pH in H2O at 27 degrees C, all in the deoxy form. Two resonances that occur between 58 and 76 ppm downfield from the water proton signal have been assigned to the hyperfine shifted proximal histidyl NH-exchangeable protons of the alpha- and beta-chains of deoxyhemoglobin. These two resonances are sensitive to the quaternary state of hemoglobin, amino acid substitutions in the alpha 1 beta 2-subunit interface and in the carboxy-terminal region of the beta-chain, and the addition of organic phosphates. The experimental results show that there are differences in the heme pockets among these four hemoglobins studied. The structural and dynamic information derived from the hyperfine shifted proximal histidyl NH-exchangeable proton resonances complement that obtained from the ferrous hyperfine shifted and exchangeable proton resonances of deoxyhemoglobin over the spectral region from 5 to 20 ppm downfield from H2O. The relationship between these findings and Perutz's stereochemical mechanism for the cooperative oxygenation of hemoglobin is discussed.     

Assignment of downfield proton resonances in purine nucleoside phosphorylase immucillin-H complex by saturation-transferred NOEs

Purine nucleoside phosphorylase (PNP) catalyzes N-ribosidic bond phosphorolysis in 6-oxypurine nucleosides and deoxynucleosides to form purine and alpha-D-phosphorylated ribosyl products. The transition state has oxacarbenium ion character with partial positive charge near C-1', ionic stabilization from the nearby phosphate anion, and protonation at N-7 of the purine. Immucillin-H (ImmH) has a protonated N-7 and resembles the transition-state charge distribution when N-4' is protonated to the cation. It binds tightly to the PNPs with a K(d) value 56 pM for human PNP. Previous NMR studies of PNP.ImmH.PO(4) have shown that the N-4' of bound ImmH is a cation and is postulated to have a significant contribution to its tight binding. Several unassigned downfield proton resonances (>11 ppm) are specific to the PNP.ImmH.PO(4) complex, suggesting the existence of strong hydrogen bonds. In this study, two of the proton resonances in this downfield region have been assigned. Using (15)N-7-labeled ImmH, a resonance at 12.5 ppm has been assigned to N-7H. The N-7H resonance is shifted downfield by only approximately 1 ppm from its position for ImmH free in aqueous solution, consistent with only a small change in the hydrogen bonding on N-7H upon binding of ImmH to PNP. In contrast, the downfield resonance at 14.9 ppm in the PNP.ImmH.PO(4) complex is assigned to N-1H of ImmH by using saturation-transferred NOE measurements on the PNP.ImmH complex. The approximately 4 ppm downfield shift of the N-1H resonance from its position for ImmH free in solution suggests that the hydrogen bonding to the N-1H in the complex has a significant contribution to the binding of ImmH to PNP. The crystal structure shows Glu201 is in a direct hydrogen bond with N-1H and to O-6 through a water bridge. In the complex with 6-thio-ImmH, the N-1H resonance is shifted further downfield by an additional 1.5 ppm to 16.4 ppm, but the relative shift from the value for 6-thio-ImmH free in solution is the same as in the ImmH complex. Since the binding affinity to hPNP for 6-thio-ImmH is decreased 440-fold relative to that for ImmH, the loss in binding energy is primarily due to the hydrogen bond energy loss at the 6-thiol.     

Proton magnetic resonance studies of carbonic anhydrase. I. Identification of histidine resonances.

Nuclear magnetic resonance (nmr) spectra of human carbonic anhydrase B recorded in deuterium oxide reveal seven discrete single proton resonances between 7 and 9 ppm downfield from sodium 2,2-dimethyl-i-silapentane-5-sulfonate. Simplification of spectra by use of Fremy's salt, comparison of peak widths at intersections, and evaluation of the results of inhibition and modification experiments permit determination of the pH dependencies of these resonances. Five of these peaks change position with increasing pH; three move upfield by approximately 95 Hz and two move downfield by 10 and 23 Hz. The first three reflect residues with pK values of 7.23, 6.98, and 6 and can be assigned to the C-2 protons of histidines. The two remaining pH dependent resonances reflect groups with pK values of 8.2 and 8.24. Their line widths and T1 values are comparable to those of the first group, and they also appear to reflect C-H protons of histidines. Despite the structural and functional similarities of the B and C isozymes of human carbonic anhydrase, few of the low field resonances appear to be common to both. Six histidine C-2 protons are observed in the C enzyme and reflect groups with pK values of approximately 7.3, 6.5, 5.7, 6.6, 6.6, and 6.4. A seventh peak contains two protons and moves upfield with increasing pH without titrating. A final resonance to low field moves downfield with increasing pH and reflects a group with a pK between 6 and 7. Its behavior resembles that of peak 1 of the human B enzyme, and it also appears to be a histidine C-H proton. This peak may reflect a conserved residue in the two isozymes that plays an important role in enzymatic function, as discussed in the following paper.     

31 P nuclear magnetic resonance studies of the interaction of pyridine nucleotide coenzymes with dehydrogenases.

31P nuclear magnetic resonance spectra of the pyrophosphate group in NAD+ and NADH were recorded in the presence of beef heart lactate dehydrogenase and rabbit muscle glyceraldehyde-3-phosphate dehydrogenase. At high lactate dehydrogenase concentrations (60 mg/ml), two NADH resonances are observed: a slowly exchanging peak which is shifted to 1.9 ppm downfield (relative to free NADH) and a rapidly exchanging peak with a downfield shift of 0.5-0.6 ppm. At lover concentrations (15 mg/ml) only the rapidly exchanging peak is observed thus indicating that the peak observed at-1.9 ppm is due to coenzyme bound to an aggregated enzyme species. With NAD+, rapid exchange and downfield shifts are observed at both enzyme and concentrations, with shifts of about 1.5 ppm and 0.6 ppm at 60 and 15 mg/ml, respectively. In the presence of glyceraldehydephosphate dehydrogenase, the results are independent of enzyme concentration, and slow exchange and upfield shifts of 0.4-0.6 ppm occur with each coenzyme. These data indicate that the environment of the pyrophosphate group of oxidized and reduced coenzyme is the same for a given dehydrogenase, but is different in one enzyme from the other. The resonances observed with glyceraldehydephosphate dehydrogenase are broader than those observed with lactate dehydrogenase. This is indicative of either shorter relaxation times with the former enzyme, or the presence of multiple, unresolved resonances.     

1H NMR of glycosaminoglycans and hyaluronic acid oligosaccharides in aqueous solution: the amide proton environment

The exchangeable amide protons of hyaluronic acid (HA) oligosaccharides and a higher-molecular-weight segment dissolved in H2O at pH 2.5 or 5.5 were examined by H NMR spectroscopy at 250 MHz. The HA segment preparation showed a single amide resonance, near the chemical shift for the amide proton of the monosaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose (beta-GlcNAc). Smaller HA oligosaccharides showed two or three separate amide proton resonances, corresponding in relative peak area to interior or end GlcNAc residues. The interior GlcNAc amide resonance occurred at the same chemical shift as the single resonance of the HA segment. For the end GlcNAc residues, linkage to D-glucuronopyranose (GlcUA) through C1 resulted in an upfield shift relative to the beta-anomer of GlcNAc, whereas linkage through C3 resulted in a downfield shift relative to the corresponding anomer of GlcNAc. These chemical-shift perturbations appeared to be approximately offsetting in the case of linkage at both positions. The amide proton vicinal coupling constant (ca. 9 Hz) was found to be essentially independent of chain length, residue position, or solution pH. These data favor a nearly perpendicular orientation for the acetamido group with respect to the sugar ring, little affected by linkage of GlcNAc to GlcUA. No evidence for the existence of a stable hydrogen bond linking the amide proton with the carboxyl(ate) oxygen of the adjacent uronic acid residue was found. The amide proton resonances for chondroitin, chondroitin 4-sulfate, and dermatan sulfate were compared to that of HA. The chemical shifts of these resonances deviated no more than 0.1 ppm from that of HA. A small dependence on the identity of the adjacent uronic acid residue was noted, based on the observation of two resonances for dermatan sulfate.     

1H NMR spectroscopy of Paracoccus denitrificans cytochrome c-550

The 1H NMR spectra of ferri- and ferro-cytochrome c-550 from Paracoccus denitrificans (ATCC 13543) have been investigated at 300 MHz. The ferri-cytochrome c-550 shows hyperfine-shifted heme methyl resonances at 29.90, 29.10, 16.70, and 12.95 ppm and a ligand methionyl methyl resonance at -15.80 ppm (pH 8 and 23 degrees C). Four pH-linked structural transitions were detected in spectra taken as a function of pH. The transitions have been interpreted as loss of the histidine heme ligand (pK less than or equal to 3), ionization of a buried heme propionate (pK = 6.3 +/- 0.2), displacement of the methionine heme ligand by a lysyl amino group (pK congruent to 10.5), and loss of the lysyl ligand (pK greater than or equal to 11.3). The temperature behavior of hyperfine-shifted resonances was determined. Two heme methyl resonances (at 16.70 and 12.95 ppm) showed downfield hyperfine shifts with increasing temperature. The cyanoferricytochrome had methyl resonances at 23.3, 20.1, and 19.4 ppm. NMR spectroscopy did not detect the formation of a complex with azide. The second-order rate constant for electron transfer between ferric and ferrous forms was determined to be 1.6 X 10(4) M-1 s-1. Heme proton resonances were assigned in both oxidation states by cross-saturation and nuclear Overhauser enhancement experiments. Spin-coupling patterns in the aromatic region of the ferro-cytochrome spectrum were investigated.     

Proton nuclear magnetic resonance saturation transfer studies of coenzyme binding to Lactobacillus casei dihydrofolate reductase

The chemical shifts of all the aromatic proton and anomeric proton resonances of NADP+, NADPH, and several structural analogues have been determined in their complexes with Lactobacillus casei dihydrofolate reductase by double-resonance (saturation transfer) experiments. The binding of NADP+ to the enzyme leads to large (0.9-1.6 ppm) downfield shifts of all the nicotinamide proton resonances and somewhat smaller upfield shifts of the adenine proton resonance. The latter signals show very similar chemical shifts in the binary and ternary complexes of NADP+ and the binary complexes of several other coenzymes, suggesting that the environment of the adenine ring is similar in all cases. In contrast, the nicotinamide proton resonances show much greater variability in position from one complex to another. The data show that the environments of the nicotinamide rings of NADP+, NADPH, and the thionicotinamide and acetylpyridine analogues of NADP+ in their binary complexes with the enzyme are quite markedly different from one another. Addition of folate or methotrexate to the binary complex has only modest effects on the nicotinamide ring of NADP+, but trimethoprim produces a substantial change in its environment. The dissociation rate constant of NADP+ from a number of complexes was also determined by saturation transfer.     

Complete structure of lipid A obtained from the lipopolysaccharides of the heptoseless mutant of Salmonella typhimurium

A highly purified monophosphoryl lipid A, TLC-3 fraction obtained from the lipopolysaccharides of the heptoseless mutant Salmonella typhimurium G30/C21 was converted to the dimethyl pentatrimethylsilyl derivative and analyzed by proton NMR spectroscopy at 400 MHz. Substantial downfield shifts of the resonances for protons at the 3- and 3'-carbons of the glucosamine disaccharide to 5.06 and 5.15 ppm, respectively, occurred from the normal range of 3.5-4.1 ppm, indicating that these two positions on the sugar rings were acylated. Significant downfield shift of the resonances for protons at the 4- and 6'-carbons did not occur, indicating the absence of acyl groups at these two positions. Since positive ion fast atom bombardment mass spectrometry previously established the presence of hydroxymyristoyl and myristoxymyristoyl esters at the reducing end and distal subunits, respectively, these acyl groups must be attached to the oxygen of the corresponding 3- and 3'-carbons of lipid A. With these results, we can now describe the complete structure of the monophosphoryl lipid A, TLC-3 from S. typhimurium.     

Conformational changes in the metal-binding sites of cardiac troponin C induced by calcium binding.

Isotope labeling of recombinant normal cardiac troponin C (cTnC3) with 15N-enriched amino acids and multidimensional NMR were used to assign the downfield-shifted amide protons of Gly residues at position 6 in Ca(2+)-binding loops II, III, and IV, as well as tightly hydrogen-bonded amides within the short antiparallel beta-sheets between pairs of Ca(2+)-binding loops. The amide protons of Gly70, Gly110, and Gly146 were found to be shifted significantly downfield from the remaining amide proton resonances in Ca(2+)-saturated cTnC3. No downfield-shifted Gly resonance was observed from the naturally inactive site I. Comparison of downfield-shifted amide protons in the Ca(2+)-saturated forms of cTnC3 and CBM-IIA, a mutant having Asp65 replaced by Ala, demonstrated that Gly70 is hydrogen bonded to the carboxylate side chain of Asp65. Thus, the hydrogen bond between Gly and Asp in positions 6 and 1, respectively, of the Ca(2+)-binding loop appears crucial for maintaining the integrity of the helix-loop-helix Ca(2+)-binding sites. In the apo- form of cTnC3, only Gly70 was found to be shifted significantly downfield with respect to the remaining amide proton resonances. Thus, even in the absence of Ca2+ at binding site II, the amide proton of Gly70 is strongly hydrogen bonded to the side-chain carboxylate of Asp65. The amide protons of Ile112 and Ile148 in the C-terminal domain and Ile36 in the N-terminal domain data-sheets exhibit chemical shifts consistent with hydrogen-bond formation between the pair of Ca(2+)-binding loops in each domain of Ca(2+)-saturated cTnC3.(ABSTRACT TRUNCATED AT 250 WORDS)     

The downfield resonances in the 1H NMR spectra of Azotobacter vinelandii and Pseudomonas putida seven-iron ferredoxins

Pseudomonas putida and Azotobacter vinelandii ferredoxins each contain one [4Fe-4S] cluster and one [3Fe-4S] cluster. Their polypeptide chains are nearly identical, differing by only 15 residues out of a total of 106. T1 measurements and temperature dependence studies of the 1H NMR spectrum of each ferredoxin demonstrate that all six resolved downfield resonances are near an iron-sulfur center. The five most downfield resonances are shown to arise from protons on cysteinyl beta-carbons by incorporation of cysteine deuterated at the beta-carbon into cell protein. The sixth peak (10.5 ppm) is shown to be a non-cysteinyl proton. This peak resolves into two resonances of approximately equal intensity at temperatures below 15 degrees or above 25 degrees C. A nuclear Overhauser effect observed between the two downfield-most resonances of A. vinelandii ferredoxin indicates that they originate from a geminal pair of beta-cysteinyl protons. An Overhauser effect observed between the resonances at 22.3 and 15.7 ppm, in conjunction with other results, implies that the resonance at 22.3 ppm arises from a beta-proton on the 3Fe-center-bound Cys16, while the resonance at 15.7 ppm arises from Cys45 beta-proton, which is bound to the 4Fe center. The five most downfield resonances are pH-dependent. The sixth peak (10.5 ppm in P. putida ferredoxin) is pH-independent. Possible origins for the observed pH dependencies are discussed.     

Assignment of resonances in the Escherichia coli 5 S RNA fragment proton NMR spectrum using uniform nitrogen-15 enrichment

The downfield proton NMR spectrum of aqueous uniformly nitrogen-15 enriched 5 S RNA fragment is presented. Selective nitrogen-15 decoupling difference proton spectroscopy revealed nitrogen-15 chemical shifts of fragment imino nitrogens. Nitrogen chemical shifts of nucleic acid guanine and uracil imino nitrogens have separate small ranges. Nitrogen-15 and proton chemical shift correlation by the heteronuclear decoupling permitted the identification of the base type of some previously unassigned imino proton resonances in the 5 S RNA fragment spectrum. Corresponding resonances in the natural isotopic abundance 5 S RNA fragment spectrum are assigned to base types by comparison with the enriched sample spectrum.     

15N-labeled tRNA. Identification of 4-thiouridine in Escherichia coli tRNASer1 and tRNATyr2 by 1H-15N two-dimensional NMR spectroscopy

Uridine is uniquely conserved at position 8 in elongator tRNAs and binds to A14 to form a reversed Hoogsteen base pair which folds the dihydrouridine loop back into the core of the L-shaped molecule. On the basis of 1H NMR studies, Hurd and co-workers (Hurd, R. E., Robillard, G. T., and Reid, B. R. (1977) Biochemistry 16, 2095-2100) concluded that the interaction between positions 8 and 14 is absent in Escherichia coli tRNAs with only 3 base pairs in the dihydrouridine stem. We have taken advantage of the unique 15N chemical shift of N3 in thiouridine to identify 1H and 15N resonances for the imino units of S4U8 and s4U9 in E. coli tRNASer1 and tRNATyr2. Model studies with chloroform-soluble derivatives of uridine and 4-thiouridine show that the chemical shifts of the protons in the imino moieties move downfield from 7.9 to 14.4 ppm and from 9.1 to 15.7 ppm, respectively; whereas, the corresponding 15N chemical shifts move downfield from 157.5 to 162.5 ppm and from 175.5 to 180.1 ppm upon hydrogen bonding to 5'-O-acetyl-2',3'-isopropylidene adenosine. The large difference in 15N chemical shifts for U and s4U allows one to unambiguously identify s4U imino resonances by 15N NMR spectroscopy. E. coli tRNASer1 and tRNATyr2 were selectively enriched with 15N at N3 of all uridines and modified uridines. Two-dimensional 1H-15N chemical shift correlation NMR spectroscopy revealed that both tRNAs have resonances with 1H and 15N chemical shifts characteristic of s4UA pairs. The 1H shift is approximately 1 ppm upfield from the typical s4U8 resonance at 14.8 ppm, presumably as a result of local diamagnetic anisotropies. An additional s4U resonance with 1H and 15N shifts typical of interaction of a bound water or a sugar hydroxyl group with s4U9 was discovered in the spectrum of tRNATyr2. Our NMR results for tRNAs with 3-base pair dihydrouridine stems suggest that these molecules have an U8A14 tertiary interaction similar to that found in tRNAs with 4-base pair dihydrouridine stems.     

Proton magnetic resonance studies of the states of ionization of histidines in native and modified subtilisins

A technique was developed to exchange the backbone -N-H protons in D2O in the native subtilisins Carlsberg and BPN (Novo) that resulted in clearly resolved proton resonances in the aromatic region of the nuclear magnetic resonance spectrum. pH titration curves for four of the five histidine C2-H resonances in subtilisin Carlsberg and five of the six in subtilisin BPN between 7.5 and 8.8 ppm downfield from 4,4-dimethyl-4-silapentane-1-sulfonic acid sodium salt provided microscopic pKa's between 6.3 and 7.2 for both sources of the enzyme at ambient (approximately 22 degrees C) probe temperature. A resonance that titrated with a pKapp of 7.35 +/- 0.05 was observed in the 1H spectra only of the diisopropylphosphoryl derivatives of the subtilisins from both sources. The 31P NMR pH titration of the same preparations under identical conditions of solvent (D2O) and temperature gave a pKapp = 7.40 +/- 0.05 of the single titratable resonance. Both observations must pertain to His-64 at the active center. A resonance smaller than the others and titrating with a pKapp of 7.2 could also be observed in the native enzymes. This resonance was assigned to the catalytic center histidine since its pK corresponded to that derived from kinetic studies. No major perturbations in the chemical shifts or the pK's derived from the pH dependence of the observed resonances were apparent in the presence of saturating concentrations of the two putative transition-state analogues phenylboronic acid and bis [3,5-(trifluoromethyl)phenyl]boronic acid and in monoisopropylphosphorylsubtilisin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformation and interaction of short nucleic acid double-stranded helices. II. Proton magnetic resonance studies on the hydrogen-bonded NH-N protons of ribosyl ApApGpCpUpU helix.

A self-complementary ribohexanucleotide, ApApGpCpUpU, was synthesized and its NH-N hydrogen-bonded protons were studied by proton magnetic resonance. At 1 degree C, 0.17 M Na+, pH 7.6 with 10 mM phosphate-0.1 mM EDTA in H2O, three proton resonances are found in the low-field region with the following chemical shifts and line widths at half-height: 13.2 ppm (80 Hz), 13.5 ppm (30 Hz), and 14.2 ppm (44 Hz). The existence of these resonances indicates the formation of a self-complementary, hydrogen-bonded duplex under these conditions. Upon elevation of temperature, these three resonances sequentially broaden and finally all disappear near 35 degrees C. Unambiguous assignments of these three resonances can be made to the terminal A(1)-U(6) pairs, interior A(2)-U(5) pairs, and to the middle G(3)-C(4) pairs. The assignments were based on (i) the differential sensitivities of the line widths of these resonances to thermal variation, as well as on (ii) a comparison of the computed chemical shifts with the observed chemical shifts. The quantitative aspects of the NH proton transfer between helix, coil, and water are discussed in relationship to the line widths of these resonances and the lifetime of the helix state. The computed chemical shifts of the NH-N resonances based on the A-RNA (or A'-RNA) model agree more closely with the observed chemical shifts than the computed values based on the B-DNA model. These results suggest that the helical duplex of A2GCU2 assumes a conformation similar to A-RNA (or A'-RNA) in aqueous solution. The results on both the NH-N resonances and the C-H resonances are summarized and discussed in terms of the helical conformation of (A2GCU2)2.     

NMR study of the phosphoryl binding loop in purine nucleotide proteins: evidence for strong hydrogen bonding in human N-ras p21

The structure of the phosphoryl binding region of human N-ras p21 was probed by using heteronuclear proton-observed NMR methods. Normal protein and a Gly-12----Asp-12 mutant protein were prepared with two amino acids labeled with 15N at their amide positions: valine and glycine, aspartic acid and glycine, and lysine and glycine. We completed the identification of amide 15NH resonances from Gly-12 and Asp-12 to the end of the phosphoryl binding domain consensus sequence (Lys-16) in protein complexed with GDP and have made tentative amide identifications from Val-9 to Ser-17. The methods used, together with initial identifications of the Gly-12 and -13 amide resonances, were described previously [Campbell-Burk, S. (1989) Biochemistry 28, 9478-9484]. The amide resonances of both Gly-13 and Lys-16 are shifted downfield below 10.4 ppm in both the normal and mutant proteins. These downfield shifts are presumed to be due to strong hydrogen bonds with the beta-phosphate oxygens of GDP.     

Cystic fibrosis transmembrane conductance regulator: solution structures of peptides based on the Phe508 region, the most common site of disease-causing DeltaF508 mutation.

Most cases of cystic fibrosis (CF), a common inherited disease of epithelial cell origin, are caused by the deletion of Phe508 located in the first nucleotide-binding domain (NBF1) of the protein called CFTR (cystic fibrosis transmembrane conductance regulator). To gain greater insight into the structure within the Phe508 region of the wild-type protein and the change in structure that occurs when this residue is deleted, we conducted nuclear magnetic resonance (NMR) studies on representative synthetic 26 and 25 amino acid peptide segments. 2D 1H NMR studies at 600 MHz of the 26-residue peptide consisting of Met498 to Ala523 in 10% DMSO, pH 4.0, at 25 degrees C show a continuous but labile helix from Gly500 to Lys522, based on both NH-NH(i,i+1) and alphaH-NH(i,i+1) NOEs. Phe508 within this helix shows only short-range (i, 相似文献