77Se NMR characterization of 77Se-labeled ovine erythrocyte glutathione peroxidase

Lambs, maintained on a selenium-deficient diet supplemented with 94 atom % Na2 27SeO3, have been used as a source of 77Se-enriched erythrocyte glutathione peroxidase. After 5 months on this diet, the percentage of selenium in the enzyme derived from the supplement had reached 88%. From each monthly bleeding of two sheep, approximately 20 mg of 77Se-enriched glutathione peroxidase could be isolated in pure form. Although attempts to observe 77Se NMR signals from the native enzyme labeled with 6,6'-[77Se]diselenobis-(3-nitrobenzoic acid) failed, due to the low solubility of the enzyme, two 77Se resonances were observed after unfolding the enzyme with 8 M urea and reaction with iodoacetamide. These resonances, at 195 and 377 ppm, were from the selenoether alkylamide derivative and from protein cross-linked selenide sulfide species, respectively. Relaxation time measurements on the selenoether at 4.7 and 9.4 teslas enabled an estimate of the chemical shift anisotropy to be made. A value of less than or equal to 262 ppm was determined. Reduction of the denatured selenide sulfide species with dithiothreitol gave an observable 77Se resonance from the Se- moiety at pH 8 and from SeH at pH 4.2. The chemical form of the selenocysteine residue in the resting state enzyme most consistent with formation of the acetamide derivative and the selenide sulfide is Se- or SeH. From the magnitudes of the estimated chemical shift anisotropies, it is predicted that direct observation of selenium in the native enzyme will be feasible if the enzyme concentration can be increased to 0.25 mM tetrameric glutathione peroxidase.     

Thermolysin-inhibitor complexes examined by 31P and 113Cd NMR spectroscopy

Complexes between phosphoramidon (N-(alpha-rhamnopyranosyloxyhydroxyphosphinyl)-L-leucyl-L-tryptoph an) and zinc thermolysin and between phosphoramidon or N-phosphoryl-L-leucineamide and 113Cd-substituted thermolysin have been examined by 31P and 113Cd NMR spectroscopy. 113Cd resonances are observed at 168 and 152 ppm for the phosphoramidon and N-phosphoryl-L-leucineamide complexes, respectively. There are large but different chemical shift anisotropy contributions to the 113Cd line widths for the two complexes, which reflect the known structural differences for the zinc-enzyme complexes. 113Cd-31P spin-spin coupling is also seen and differs for the two cadmium complexes, being larger, 28 Hz, for the bidentate N-phosphoryl-L-leucineamide ligand than for the monodentate phosphoramidon, 16 Hz. Large changes in chemical shift, 7.5-10.9 ppm, are seen for the 31P resonances of the inhibitors upon binding to the enzyme reflecting direct phosphoryl-metal ligation. Chemical shift anisotropy is the dominant relaxation mechanism for the 31P nuclei at 9.4 T, while the dipole-dipole contribution seems to be unaffected by a change of solvent from H2O to D2O.     

Transfer RNA structure by carbon NMR: C2 of adenine, uracil and cytosine.

Fourier transform 13C NMR spectra of E. coli tRNA enriched on 13C in either position 2 of adenine (60 atom % 13C) or in position 2 of uracil (82%) and cytosine (63%) were taken at 25.16 MHz over the temperature range 10 degrees - 76 degrees. For C2 of adenine the peak as initially 5 ppm wide, but narrowed to 0.5 ppm as the molecule unfolded. C2 of uracil displayed behavior similar to that of adenine while the cytosine peak, initially relatively narrow at low temperature, sharpened less dramatically. Comparison of spectra at 26.16 MHz and 67.9 MHz showed that the peak widths for folded tRNA were determined largely by chemical shift non-equivalence. T2 T2 measurements suggested that intrinsic line widths of most cytosine C2 peaks were 4 Hz and 2-3 Hz for uracil. Adenine C2 with a directly bonded proton had resonances of about 40 Hz line width. T1 values were measured for C2 of adenine and the ribose carbons of tRNA. Consideration of dipolar relaxation and chemical shift anisotrophy led to a calculated rotational correlation time of 1.6 +/- 0.4 x 10(-8) sec for the adenines and 1.3 +/- 0.3 x 10(-8) sec for the ribose carbons.     

Demonstration of the feasibility of observing nuclear magnetic resonance signals of 77Se covalently attached to proteins

Previous 77Se NMR relaxation time studies established the utility of 77Se NMR spectroscopy in studying low molecular weight (less than 500) selenium-containing molecules. Since the spin rotation and chemical shift anisotrophy mechanisms contributed significantly to the 77Se spin-lattice relaxation in these compounds, it was questionable as to whether the latter mechanism would be efficient enough to enable 77Se resonances to be observed in a reasonable period in high molecular weight selenobiomolecules. Thus, to address this problem, disulfide bonds of ribonuclease-A and lysozyme were reductively cleaved under denaturing conditions, and the resulting 7-8 sulfhydryl groups were treated with a new sulfhydryl group reagent containing selenium, 6,6'-diselenobis(3-nitrobenzoic acid), to give proteins containing covalently attached selenium in the form of selenenyl sulfides. The observation of high resolution 77Se NMR spectra of these proteins under denaturing conditions was accomplished. Five to six 77Se NMR resonances, which fell in a chemical shift range of 14-15 ppm, were observed for each protein and are compared to the chemical shifts of several model selenenyl sulfides derived from cysteine.     

Deuterium nuclear magnetic resonance of deuterium-labeled diacetyldeuterohemin incorporated into sperm whale myoglobin.

The heme derivative 2,4-diacetyldeuterohemin deuterated in the methyl groups of the acetyl moieties was reconstituted with sperm whale apomyoglobin and the two labeled methyl groups in the protein environment were observed by deuterium nuclear magnetic resonance spectroscopy. The results were compared to the free hemin form as the dimethyl ester in chloroform and in a pyridine-water mixture, as well as in the zinc complex form. Under most conditions the two methyl resonances overlie each other to a large degree. Resonance width at half-height is of the order of 25 Hz for the protein and approximately one-third as much for the free hemin at 16 degrees and is little affected by conversion to paramagnetic derivatives. Chemical shifts for the oxy- and carbonmonoxymyoglobins are very similar. In cyanoferrimyoglobin a positive pseudo-contact contribution of 3.04 ppm was computed to explain a relative upfield shift offset in part by a small negative contact shift contribution. The cyanoferrimyoglobin resonance was sensitive to the presence of phosphate buffer as well as to cyclopropane. The aquoferrimyoglobin form shows distinct resonances for the two methyl groups, with the downfield resonance considerably broadened. The expected effects of temperature on chemical shift were observed, the paramagnetic derivatives showing an effect and carbonmonoxymyoglobin showing none. The relaxation behavior was gauged from the line widths and from measurements of spin-lattice relaxation time, T1. The effective rotational correlation time is of the order of 50 ps for the liganded myoglobin forms. The temperature dependence of the line widths may imply an increased retational freedom with increasing temperature. The broadening observed in the aquoferrimyoglobin case is indicative of restricted internal rotational motion of one of the methyl groups. The method is suitable for probing the more mobile structures in proteins and retains its value in the neighborhood of paramagnetic centers.     

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.     

High pressure NMR studies of hemoproteins. The effect of pressure on the quaternary structure of hemoglobin

Proton NMR spectra for nitrosyl-, aquomet- and deoxy des-Arg(α141)-hemoglobin in H₂O were studied at high pressures up to 1400 atm with attention to the exchangeable proton resonances due to the intra- and intersubunit hydrogen bonds. For aquomethemoglboin, the T state marker signal at 6.4 ppm is insensitive to pressure while the R state marker signal at 6.0 ppm exhibits progressive upfield shift upon pressurization. For nitrosylhemoglobin, the T state signals at 9.6 and 6.5 ppm decrease their intensities upon pressurization while the R state marker signal at 6.0ppm remains unchanged. Pressure-induced spectral changes for some of exchangeable resonances are also encountered for deoxy des-Arg(α141)-hemoglobin while the R and T quaternary structural indicators at 6.0 and 9.4 ppm are insensitive to pressure. These pressure-induced spectral changes for these hemoglobin derivatives are significantly distinguished from those associated with the R-T transition induced by addition of IHP or by variatiuon of pH. It is therefore concluded that pressure induces subtle quaternary structural changes in these hemoglobin derivatives without causing the R-T transition.     

13C NMR spectroscopy of labeled pyridoxal 5'-phosphate. Model studies, D-serine dehydratase, and L-glutamate decarboxylase.

Pyridoxal 5'-phosphate labeled to the extent of 90% with 13C in the 4' (aldehyde) and 5' (methylene) positions has been synthesized. 13C NMR spectra of this material and of natural abundance pyridoxal 5'-phosphate are reported, as well as 13C NMR spectra of the Schiff base formed by reaction of pyridoxal 5'-phosphate with n-butylamine, the secondary amine formed by reduction of this Schiff base, the thiazolidine formed by reaction of pyridoxal 5'-phosphate with cysteine, the hexahydropyrimidine formed by reaction of pyridoxal 5'-phosphate with 1,3-diaminobutane, and pyridoxamine 5'-phosphate. The range of chemical shifts for carbon 4' in these compounds is more than 100 ppm, and thus this chemical shift is expected to be a sensitive indicator of structure in enzyme-bound pyridoxal 5'-phosphate. The chemical shift of carbon 5', on the other hand, is insensitive to these structure changes. 13C NMR spectra have been obtained at pH 7.8 and 9.4 for D-serine dehydratase (Mr = 46,000) containing natural abundance pyridoxal 5'-phosphate and containing 13C-enriched pyridoxal 5'-phosphate. The enriched material contains two new resonances not present in the natural abundance material, one at 167.7 ppm with a linewidth of approximately 24 Hz, attributed to carbon 4' of the Schiff base in the bound coenzyme, and one at 62.7 Hz with a linewidth of approximately 48 Hz attributed to carbon 5' of the bound Schiff base. A large number of resonances due to individual amino acids are assigned. The NMR spectrum changes only slightly when the pH is raised to 9.4. The widths of the two enriched coenzyme resonances indicate that the coenzyme is rather rigidly bound to the enzyme but probably has limited motional freedom relative to the protein. 13C NMR spectra have been obtained for L-glutamate decarboxylase containing natural abundance pyridoxal 5'-phosphate and 13C-enriched pyridoxal 5'-phosphate. Under conditions where the two enriched 13C resonances are clearly visible in D-serine dehydratase, no resonances are visible in enriched L-glutamate decarboxylase, presumably because the coenzyme is rigidly bound to the protein and the 300,000 molecular weight of this enzyme produces very short relaxation times for the bound coenzyme and thus very broad lines.     

Detection and characterization of hyperfine-shifted resonances in the proton nuclear magnetic resonance spectrum of Anabaena 7120 ferredoxin at high magnetic fields

This paper presents previously unobserved signals in the 1H NMR spectra of oxidized and reduced [2Fe-2S]-ferredoxin from Anabaena 7120 detected at 400, 500, and 600 MHz. The signals shifted to low field exhibited longitudinal relaxation (T1) values in the range of 100-400 microseconds and line widths in the range of 1-10 kHz (at 400 MHz), and the chemical shifts of all signals showed strong temperature dependence. Although the line widths were smaller at lower magnetic fields, the resolution was better at higher magnetic fields. In the oxidized state, a broad signal was detected at 37 ppm, which corresponds to at least 6 protons, and whose chemical shift exhibits positive temperature dependence. This signal also was found in oxidized ferredoxin reconstituted in 2H2O, which excludes the signal as arising from solvent-exchangeable amide protons. In the reduced state, four signals detected between 90 and 140 ppm exhibited negative temperature dependence. These consisted of two pairs of signals, each pair having one component with half the linewidth of the other. On the basis of their chemical shifts, linewidths, longitudinal relaxation properties, and temperature dependence we assigned these resonances to four of the beta hydrogens of the ligated cysteines. Two solvent-exchangeable hyperfine-shifted signals were found in the reduced state; these are located upfield of the diamagnetic region. The low-field hyperfine resonances of half-reduced ferredoxin in the presence of sodium dithionite showed a self electron transfer exchange rate that was slow on the NMR scale as observed earlier (Chan, T., and Markley, J. L. (1983) Biochemistry 22, 5982-5987), but the exchange rate was accelerated in the presence of methyl viologen.     

19F NMR investigation of molecular motion and packing in sonicated phospholipid vesicles

Dimyristoylphosphatidylcholine (DMPC) labeled with a C19F2 group in the 4-, 8-, or 12-position of the 2-acyl chain has been investigated in sonicated unilamellar vesicles (SUV) by fluorine-19 nuclear magnetic resonance (NMR) at 282.4 MHz from 26 to 42 degrees C. The 19F NMR spectra exhibit two overlapping resonances with different line widths. Spin-lattice relaxation time measurements have been performed in both the laboratory frame (T1) and the rotating frame (T1 rho) in order to investigate the packing and dynamics of phospholipids in lipid bilayers. Quantitative line-shape and relaxation analyses are possible by using the experimental chemical shift anisotropy (delta nu CSA) and the internuclear F-F vector order parameter (SFF) values obtained from the 19F powder spectra of multilamellar liposomes. The following conclusions can be made: The 19F chemical shift difference between the inside and outside leaflets of SUV can be used to monitor the lateral packing of the phospholipid in the two SUV monolayers. The hydrocarbon chains in the outer layer are found to be more tightly packed than those of the inner one, and the differences between them become smaller near the chain terminals. The effective correlation time [(1-4) x 10(-7) s] obtained from either the motional narrowing of the line widths or off-resonance T1 rho measurements is shorter than that estimated from the Stokes-Einstein diffusion model (10(-6) s), on the basis of a hydrodynamic radius of 110 A for SUV.(ABSTRACT TRUNCATED AT 250 WORDS)     

31P NMR relaxation measurements of the phosphate backbone of a double stranded hexadeoxynucleotide in solution: determination of the chemical shift anisotropy

The five phosphates of the deoxynucleotide d(CpGpTpApCpG)₂ have been assigned by two-dimensional heteronuclear NMR spectroscopy. The chemical shift anisotropy and correlation time of each phosphate group has been determined from measurements of the spin-lattice, spin-spin relaxation rate constants and the ³¹P-{¹H} nuclear Overhauser enhancement (NOE) at three magnetic field strengths (4.7 T, 9.4 T, and 11.75 T) and two temperatures (288 K and 298 K). As expected, the relaxation data require two mechanisms to account for the observed rate constants, i.e. dipole-dipole and chemical shift anisotropy. At 9.4 T and 11.75 T, the latter mechanism dominates the relaxation, leading to insignificant NOE intensities. The correlation time, chemical shift anisotropy and effective P-H distance were obtained from least-squares fitting to the data. Comparison of the fitted value for the correlation time with that obtained from ¹H measurements shows that the molecule behaves essentially as rigid rotor on the nanosecond timescale. Large amplitude motions observed in long segments of DNA are due to bending motions that do not contribute significantly to relaxation in short oligonucleotides.Abbreviations CSA chemical shift anisotropy - NOE nuclear Overhauser enhancement Offprint requests to: A. N. Lane     

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.     

Multinuclear magnetic resonance studies of metal ion binding sites of phosphoglucomutase

Metal binding at the activating site of rabbit muscle phosphoglucomutase has been studied by 31P, 7Li, and 113Cd NMR spectroscopy. A 7Li NMR signal of the binary Li+ complex of the phosphoenzyme was not observed probably because of rapid transverse relaxation of the bound ion due to chemical exchange with free Li+. The phosphoenzyme-Li+-glucose 6-phosphate ternary complex is more stable, kinetically, and yields a well-resolved peak from bound Li+ at -0.24 ppm from LiCl with a line width of 5 Hz and a T1 relaxation time of 0.51 +/- 0.07 s at 78 MHz. When glucose 1-phosphate was bound, instead, the chemical shift of bound 7Li+ was -0.13 ppm; and in the Li+ complex of the dephosphoenzyme and glucose bisphosphate a partially broadened 7Li+ peak appeared at -0.08 ppm. Thus, the bound metal ion has a somewhat different environment in each of these three ternary complexes. The 113Cd NMR signal of the binary Cd2+ complex of the phosphoenzyme appears at 22 ppm relative to Cd(ClO4)2 with a line width of 20 Hz at 44.4 MHz. Binding of substrate and formation of the Cd2+ complex of the dephosphoenzyme and glucose bisphosphate broaden the 113Cd NMR signal to 70 Hz and shift it to 75 ppm. The 53 ppm downfield shift upon the addition of substrate along with 1H NMR data suggests that one oxygen ligand to Cd2+ in the binary complex is replaced by a nitrogen ligand at some intermediate point in the enzymic reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

A proton nuclear magnetic resonance study of the quaternary structure of human homoglobins in water.

Proton nuclear magnetic resonance spectra of human hemoglobins in water reveal several exchangeable protons which are indicators of the quaternary structures of both the liganded and unliganded molecules. A comparison of the spectra of normal human adult hemoglobin with those of mutant hemoglobins Chesapeake (FG4alpha92 Arg yields Leu), Titusville (G1alpha94 Asp yields Asn), M Milwaukee (E11beta67 Val yields Glu), Malmo (FG4beta97 His yields Gln), Kempsey (G1beta99 Asp yields Asn), Yakima (G1beta99 Asp yields His), and New York (G15beta113 Val yields Glu), as well as with those of chemically modified hemoglobins Des-Arg(alpha141), Des-His(beta146), NES (on Cys-beta93)-Des-Arg(alpha141), and spin-labeled hemoglobin [Cys-beta93 reacted with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide], suggests that the proton in the important hydrogen bond between the tyrosine at C7alpha42 and the aspartic acid at G1beta99, which anchors the alpha1beta2 subunits of deoxyhemoglobin (a characteristic feature of the deoxy quaternary structure), is responsible for the resonance at -9.4 ppm from water at 27 degrees. Another exchangeable proton resonance which occurs at -6.4 ppm from H2O is a spectroscopic indicator of the deoxy structure. A resonance at -5.8 ppm from H2O, which is an indicator of the oxy conformation, is believed to originate from the hydrogen bond between the aspartic acid at G1alpha94 and the asparagine at G4beta102 in the alpha1beta2 subunit interface (a characteristic feature of the oxy quaternary structure). In the spectrum of methemoglobin at pH 6.2 both the -6.4- and the -5.8ppm resonances are present but not the -9.4-ppm resonance. Upon the addition of inositol hexaphosphate to methemoglobin at pH 6.2, the usual resonance at -9.4 ppm is shifted to -10 ppm and the resonance at 6.4 ppm is not observed. In the spectrum of methemoglobin at pH greater than or equal to 7.6 with or without inositol hexaphosphate, the resonance at -5.8 ppm is present, but not those at -10 and -6.4 ppm, suggesting that methemoglobin at high pH has an oxy-like structure. Two resonances (at -8.2 and -7.3 ppm) which remain invariant in the two quaternary structures could come from exchangeable protons in the alpha1beta1 subunit interface and/or other exchangeable protons in the hemoglobin molecule which undergo no conformational changes during the oxygenation process. These exchangeable proton resonances serve as excellent spectroscopic probes of the quaternary structures of the subunit interfaces in studies of the molecular mechanism of cooperative ligand binding to hemoglobin.     

Evidence for extensive anisotropic local motions in a small enzyme using a new method to determine NMR cross-correlated relaxation rates in the absence of resolved scalar coupling

Transverse ¹³CO-¹HN (dipole-dipole)/¹³CO (CSA) cross-correlated relaxation rates were measured for the ¹³CO resonances of the protein ribonuclease Binase from Bacillus intermedius (12.3 kDa). This was carried out with a novel E.COSY-type triple-resonance experiment, which allows the measurement of cross-correlated transverse relaxation rate from multiplet effects in the absence of resolved scalar coupling. The ¹³CO-¹HN (dipole-dipole)/¹³CO (CSA) cross-correlated relaxation rates were determined with an average precision of ±5% and cover a range of values between –1.5 and +0.6 Hz. The average (–0.44 Hz) is to be compared with the computed value of –0.83 Hz for this interaction. Mechanisms that potentially can cause the average to be smaller than the theoretical value and the unexpected large spread in observed values are discussed. It is suggested that large contributions to the variations are due to large amplitude local anisotropic motions.     

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.     

Rotational motions in myoglobin assessed by carbon 13 relaxation measurements at two magnetic field strengths.

Proton-decoupled Fourier transform nuclear magnetic resonance spectroscopy of natural abundance 13C was used to obtain spectra of cyanoferrimyoglobin of sperm whale (Physeter catadon) at 14.1 and 23.5 kG. Comparison of the spin lattice relaxation times at these two field strengths allowed the unambiguous assignment of a rotational correlation time of 22 plus or minus 5 ns for the alpha carbon resonances. The spin lattice relaxation time value for a major band attributable to aromatic carbon atoms also corresponded to a single correlation time, attributable to over-all tumbling of the molecule. Certain narrower resonances reflect other modes of rotational motion in addition to the over-all tumbling. Observations of nuclear Overhauser enhancement and line widths accord with these conslusions.     

Cadmium-113 and magnesium-25 NMR study of the divalent metal binding sites of isocitrate dehydrogenases from pig heart

The metal activator sites of NAD⁺-dependent and NADP⁺-dependent isocitrate dehydrogenases from pig heart have been probed using ¹¹³Cd- and ²⁵Mg-NMR. In the presence of isocitrate and ADP, a broad resonance for cadmium bound to NAD-dependent isocitrate dehydrogenase was observed ( −8 ppm) arising from exchange with isocitrate (−20 ppm) and/or ADP (27 ppm) complexes. The Cd shift with ADP suggests interaction of the metal with the nucleotide ring nitrogen. Increasing shifts with excess ADP are indicative of macrochelate formation. ²⁵Mg-NMR demonstrates that, unlike manganese, magnesium has a similar dissociation constant (1.8 mM) from NADP-dependent isocitrate dehydrogenase as from the enzyme-isocitrate complex (1.1 mM). The extrapolated line width of bound magnesium increases from 674 Hz in the binary complex to 10 200 Hz in the ternary complex. The quadrupole coupling constant, calculated from relaxation rates, is larger in the ternary complex. indicative of greater distortion in the magnesium coordination sphere. The line widths of magnesium complexed to NAD-dependent isocitrate dehydrogenase are broader, as expected for the larger octamer. ¹¹³Cd- and ²⁵Mg-NMR both show that the metal sites have anisotropic octahedral symmetry. ²⁵Mg relaxation rates yield correlation times corresponding to motions of a domain with motion independent of the enzyme multimers.     

Carbon-13 nuclear magnetic resonance studies on tobacco mosaic virus and its protein.

Fourier transform nuclear magnetic resonance studies on 12% 13C-enriched tobacco mosaic virus (TMV) and its rod-like protein oligomers in solution with molecular weights up to 42 X 10(6) are reported. In the virus approximately 17% of the carbons of the protein subunit have line widths of less than or equal to 300 Hz and T1 less than or equal to 1 s and are concluded to be mobile with more than one degree of freedom of internal rotation about a carbon--carbon bond. In the rodlike polymer of TMV protein at pH 5.3, 30% of the carbons are mobile, which implies rotational motions about carbon--carbon bonds and/or motions of the protein subunits within the polymer. The presence of internal mobility is supported by the observation that 20% of the carbons in the double disklike oligomer show decreasing line width upon increasing temperature; the remaining resonances have line widths which are temperature independent during the double disklike polymerization process. Since the molecular weight of TMV protein polymers increases with increasing temperature, this demonstrates that all nuclei within the double dislike oligomer are mobile. NMR and X-ray data on the double disklike polymer reveal differences with respect to internal mobility.     

13C NMR studies of porphobilinogen synthase: observation of intermediates bound to a 280,000-dalton protein

13C NMR has been used to observe the equilibrium complex of [4-13C]-5-aminolevulinate ([4-13C]ALA) bound to porphobilinogen (PBG) synthase (5-aminolevulinate dehydratase), a 280,000-dalton protein. [4-13C]ALA (chemical shift = 205.9 ppm) forms [3,5-13C]PBG (chemical shifts = 121.0 and 123.0 ppm). PBG prepared from a mixture of [4-13C]ALA and [15N]ALA was used to assign the 121.0 and 123.0 ppm resonances to C5 and C3, respectively. For the enzyme-bound equilibrium complex formed from holoenzyme and [4-13C]ALA, two peaks of equal area with chemical shifts of 121.5 and 127.2 ppm are observed (line widths approximately 50 Hz), indicating that the predominant species is probably a distorted form of PBG. When excess free PBG is present, it is in slow exchange with bound PBG, indicating an exchange rate of less than 10 s-1, which is consistent with the turnover rate of the enzyme. For the complex formed from [4-13C]ALA and methyl methanethiosulfonate (MMTS) modified PBG synthase, which does not catalyze PBG formation, the predominant species is a Schiff base adduct (chemical shift = 166.5 ppm, line width approximately 50 Hz). Free ALA is in slow exchange with the Schiff base. Activation of the MMTS-modified enzyme-Schiff base complex with 113Cd and 2-mercaptoethanol results in the loss of the Schiff base signal and the appearance of bound PBG with the same chemical shifts as for the bound equilibrium complex with Zn(II) enzyme. Neither splitting nor broadening from 113Cd-13C coupling was observed.