Correlations between fluorine-19 nuclear magnetic resonance chemical shift and the secondary and tertiary structure of 5-fluorouracil-substituted tRNA.

To complete assignment of the 19F nuclear magnetic resonance (NMR) spectrum of 5-fluorouracil-substituted Escherichia coli tRNA(Val), resonances from 5-fluorouracil residues involved in tertiary interactions have been identified. Because these assignments could not be made directly by the base-replacement method used to assign 5-fluorouracil residues in loop and stem regions of the tRNA, alternative assignment strategies were employed. FU54 and FU55 were identified by 19F homonuclear Overhauser experiments and were then assigned by comparison of their 19F NMR spectra with those of 5-fluorouracil-labeled yeast tRNA(Phe) mutants having FU54 replaced by adenine and FU55 replaced by cytosine. FU8 and FU12, were assigned from the 19F NMR spectrum of the tRNA(Val) mutant in which the base triple G9-C23-G12 substituted for the wild-type A9-A23-FU12. Although replacement of the conserved U8 (FU8) with A or C disrupts the tertiary structure of tRNA(Val), it has only a small effect on the catalytic turnover number of valyl-tRNA synthetase, while reducing the affinity of the tRNA for enzyme. Analysis of the 19F chemical shift assignments of all 14 resonances in the spectrum of 5-fluorouracil-substituted tRNAVal indicated a strong correlation to tRNA secondary and tertiary structure. 5-Fluorouracil residues in loop regions gave rise to peaks in the central region of the spectrum, 4.4 to 4.9 parts per million (p.p.m.) downfield from free 5-fluorouracil. However, the signal from FU59, in the T-loop of tRNA(Val), was shifted more than 1 p.p.m. downfield, to 5.9 p.p.m., presumably because of the involvement of this fluorouracil in the tertiary interactions between the T and D-loops. The 19F chemical shift moved upfield, to the 2.0 to 2.8 p.p.m. range, when fluorouracil was base-paired with adenine in helical stems. This upfield shift was less pronounced for the fluorine of the FU7.A66 base-pair, located at the base of the acceptor stem, an indication that FU7 is only partially stacked on the adjacent G49 in the continuous acceptor stem/T-stem helix. An unanticipated finding was that the 19F resonances of 5-fluorouracil residues wobble base-paired with guanine were shifted 4 to 5 p.p.m. downfield of those from fluorouracil residues paired with A. In the 19F NMR spectra of all fluorinated tRNAs studied, the farthest downfield peak corresponded to FU55, which replaced the conserved pseudouridine normally found at this position.

Effect of nucleoside modifications on the structure and thermal stability of Escherichia coli valine tRNA.

Transfer RNA transcribed in vitro lacks the base modifications found in native tRNA. To understand the effect of base modifications on the structure of tRNA, the downfield region of the 1H NMR spectrum of in vitro transcribed E coli tRNAVal in aqueous phosphate buffer in the presence of excess Mg2+ was investigated. The resonances of all imino protons involved in hydrogen bonds in the helical stem regions and in tertiary interactions were assigned using two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) and one-dimensional difference nuclear Overhauser effect (NOE) methods. In addition, some aromatic C2 and C8 proton resonances as well as one amino proton resonance were assigned. The chemical shifts of the assigned resonances of unmodified E coli tRNAVal were compared with those of the native tRNA molecule under similar solution conditions. The similarity of the NMR data for unmodified and modified tRNA indicates that the in vitro transcribed tRNA has nearly the same solution structure as the native molecule in the presence of excess Mg2+. The only significant differences were the chemical shifts of resonances corresponding to protons in (or interacting with) bases, indicating the possibility of local structural perturbations. The thermal stability of E coli modified and unmodified tRNAVal in the presence of Mg2+ was also investigated by analyzing the temperature dependence of the imino proton spectra. Several tertiary interactions involving modified nucleosides in native E coli tRNAVal are less stable in the absence of base modifications.

Characterization of the pH titration shifts of ribonuclease A by one- and two-dimensional nuclear magnetic resonance spectroscopy.

One- and two-dimensional 1H NMR experiments were used to determine the chemical shifts of resonances arising from 29 residues of RNase A in H2O at 17 pH values ranging from 1.2 to 7.9 Nearly all resonances displaying pH-induced changes in chemical shift greater than 0.1 ppm were monitored. Individual plots of the chemical shift as a function of pH were fit by nonlinear least squares methods to Henderson-Hasselbalch models yielding pK alpha values which were then analyzed using a set of criteria to determine their reliability. The criteria included statistics from the curve fitting analysis as well as the distance of the reporter proton to ionizing groups. Only the most reliable pK alpha values were assigned to specific ionizing groups within RNase A based upon the proximity of the reporter proton to the ionizing group as determined from the X-ray crystal structure. Only 2 of the 15 groups expected to undergo ionization within the pH range investigated could not be assigned pK alpha values within the highest two levels of reliability. Of the 11 carboxylate groups, 5 have pK alpha values less than 3.0. Many of the low pK alpha values can be interpreted as resulting from favorable hydrogen bonds between the carboxylate group and other moieties within the protein. The pK alpha values for the four histidine residues are similar to earlier literature reports. Two resonances underwent particularly large pH-induced shifts of approximately 2.3 ppm and corresponded to nitrogen-bound protons involved in hydrogen bonds with carboxylate groups.

Nucleoside modifications stabilize Mg2+ binding in Escherichia coli tRNA(Val): an imino proton NMR investigation.

The structures of in vitro transcribed Escherichia coli tRNA(Val), which lacks base modifications, and the native tRNA, which contains them, are very similar in the presence of excess Mg2+ (Kintanar, Yue, and Horowitz, unpublished results). To further probe the effects of base modifications on the structure of tRNA, the Mg2+ ion dependence of the downfield region of the 1H NMR spectrum of in vitro transcribed E. coli tRNA(Val) in aqueous phosphate buffer was investigated. The spectra indicate a remarkable conformational change in unmodified E. coli tRNA(Val) coincident with binding or release of Mg2+. Assignment of the imino proton resonances in the low Mg2+ form of the tRNA transcript allows a detailed description of the conformational change. There is near total disruption of the D stem and tertiary interactions in the absence of bound Mg2+. A new strong interaction between the U67-A6 base pair and the G50-U64 wobble pair is observed, indicating a substantial structural rearrangement at the junction of the acceptor and T stems. The binding constants of the strong Mg2+ binding sites in the D loop and near the D stem in unmodified tRNA(Val) are at least 2 orders of magnitude less than in tRNAVal containing base modifications. The metal ion binding site in the anticodon loop is somewhat stronger than metal ion binding sites in the D loop and stem in unmodified tRNA(Val), but it is still weaker than all strong Mg2+ binding sites in native tRNA(Val). Thus, one role of the base modifications found in tRNA is to stabilize or strengthen the Mg2+ binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Human telomeric C-strand tetraplexes.

Telomeric C-strand sequences form non-Watson-Crick base-paired structures in supercoiled plasmids and in oligonucleotides at low pH. Here we examine oligonucleotides composed of 2 or 4 repeats of the human telomeric C-strand sequence d(CCCTAA)n. At low pH, the 2-repeat molecule forms a dimer which exhibits H1'-H1' nuclear Overhauser effects (NOEs) between stacked CC+ base pairs. These NOEs are characteristic of the i-motif, which is a tetraplex composed of two intercalated CC+ duplexes. The 4-repeat molecule forms an intramolecular monomeric structure at low pH, suggesting that four contiguous cytosine tracts fold into a CC+ intercalated tetraplex. These unusual structures may be relevant to the formation of guanine tetraplexes by complementary G-rich sequences. They may also provide a general mechanism for self-recognition by nucleic acids.

Deuterium nuclear magnetic resonance spectroscopic study of the fluorescent probe diphenylhexatriene in model membrane systems.

We have investigated the deuterium (2H) nuclear magnetic resonance (NMR) spectra of two 2H-labeled fluorescence probes (trans,trans,trans-1,6-diphenylhexa-1,3,5-trienes, DPHs) incorporated into model lipid bilayer membrane systems at various temperatures. The membranes consisted of multilamellar bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) containing varying concentrations of cholesterol. The conventional one-order parameter approach often used in the analysis of the NMR data of lipid membranes does not explain the observed temperature variations of the spectral features. Consistent with the molecular symmetry, the results have thus been analyzed in terms of an ordering matrix with more than one independent element. The molecular order parameter (SNMR), the order along the long molecular axis, in the pure lipid system varies from 0.49 to 0.26 as the temperature is increased from 25 to 57 degrees C. These values are somewhat larger than the order parameters obtained from fluorescence depolarization (SFLU) on sonicated DMPC vesicles. Such discrepancies probably arise from the looser packing of the sonicated vesicles. Addition of cholesterol to the model membranes causes the order parameter of the probe molecules to increase. At 35 degrees C, SNMR increases from 0.38 (with no cholesterol) to 0.92 (in the presence of 50 mol % cholesterol). These values are about 10% larger than those obtained from fluorescence depolarization studies on sonicated vesicles. The SNMR for DPH are somewhat larger than those obtained in earlier NMR studies of 2H-labeled cholesterol. However, they compare well with those obtained for 2H-labeled DMPC.(ABSTRACT TRUNCATED AT 250 WORDS)

Two-dimensional NMR investigation of a bent DNA fragment: assignment of the proton resonances and preliminary structure analysis.

The resonances of all the non-exchangeable protons (except 5'H and 5"H) of d(CGAAAAATCGG) + d(CCGATTTTTCG), a putatively bent DNA duplex, have been assigned using 1H two-dimensional nuclear magnetic resonance methods. The nuclear Overhauser effect data indicate an overall B-form structure for this double-helical DNA undecamer. However, several features of the NMR data such as some unusually weak C8/C6 proton to C1' proton NOE cross-peaks, the presence of relatively intense C2H to C1'H NOE cross-peaks, and unusual chemical shifts of some 2", 2', and 1' protons suggest a substantial perturbation of the helix structure at the junctions and along the length of the tract of A residues. These structural deviations are considered in terms of models of DNA bending.

Dynamics of amino acid side chains in membrane proteins by high field solid state deuterium nuclear magnetic resonance spectroscopy. Phenylalanine, tyrosine, and tryptophan.

We have obtained the first deuterium NMR spectra of individual types of aromatic amino acids in a defined membrane protein, bacteriorhodopsin, in the photosynthetic purple membrane of Halobacterium halobium R1. Isotopic labeling and high field (8.5 Tesla) operation permitted relatively rapid data acquisition at a variety of temperatures. At the temperature of growth (37 degrees C), we find that all 7 tryptophan residues are rigid on the time scale of the NMR experiment (approximately 10(-5) s), except for likely librational motions of approximately 10 degrees amplitude. By contrast, nearly all (9 +/- 2) of the 11 tyrosines and (13 +/- 2) 13 phenylalanines undergo rapid (greater than 10(5) s-1) 2-fold rotational flips about C gamma-C zeta, causing formation of line shapes dominated by effectively axially asymmetric (asymmetry parameter eta = 0.66) deuteron electric field gradient tensors. On cooling the phenylalanine- and tyrosine-labeled samples to approximately -30 degrees C, all such motions are frozen out, i.e. occur at rates less than 10(4) s-1, and axially symmetric (eta approximately 0.05) line shapes are observed. At T greater than 91 degrees C, phenylalanine-, tyrosine-, and tryptophan-labeled membrane spectra undergo dramatic narrowing to an isotropic line of approximately 4-9 kHz width. This transition is a reflection of the loss of tertiary structure in the membrane protein with resultant fast unrestricted motion of these aromatic side chains, and is only partly reversible. These results, in conjunction with those obtained using [gamma-2H6]valine-labeled bacteriorhodopsin (Kinsey, R. A., Kintanar, A., Tsai, M-D., Smith, R. L., Janes, N., and Oldfield, E. (1981) J. Biol. Chem., 256, 4146-4149) indicate the rather rigid nature of amino acid side chains in the H. halobium purple membrane, the principal fast lage amplitude motions being methyl group rotation and discontinuous benzene ring "flipping."

Sequence effects on local DNA topology.

Nuclear Overhauser effect-derived distances between adenine H2 protons and anomeric H1' protons on the same strand or on the complementary strand are presented for several different DNA duplexes. The cross-strand (n)AH2 to (m + 1)H1' distances [designated as x, where (n) and (m) are complementary residues] vary by up to 1 A depending on the sequence. In all possible A-containing pyrimidine-purine steps (CA, TG, and TA), x is greater than 4.5 A. In GA steps, x varies within rather wide limits in the range 3.8-4.5 A, whereas in AA steps the lower limit is 3.7 A and the upper limit is approximately 4.2 A. In purine-purine steps, x is affected by at least three factors: (i) adjacent pyrimidine-purine steps at the 5' end [e.g., YRA sequences (where Y = T or C and R = G or A)], or a pyrimidine-purine step at the 3' end of the pyrimidine-pyrimidine step on the complementary strand, cause x to increase, (ii) an AT step at the 3' end of a purine-purine step (e.g., RAT) causes x to decrease, and (iii) substitution of bases at the next-nearest neighbor position leads to changes in x at GA and AA steps. The latter factor seems to be due to a cooperative effect arising from formation of the "anomalous" B' structure when the substitution produces an AnTm tract (which always produces a decrease in x). The data indicate that (n)AH2-(n + 1)H1' distances on the same strand (designated as s) are also sequence dependent. Thus on AA steps, neighboring substitutions produce the same effect on s as on the cross-strand x distances. The results lead to the ability to predict changes in AH2-H1' distances depending on the DNA sequence. By using high-resolution x-ray B-type structures as a set of allowable B conformations, a very good correlation was found between x and the minor groove width parameters P-P or H1'-H1'. Thus, the x distances are a direct probe of the minor groove width in B-type DNA, and changes in this distance therefore reflect changes in the minor groove width. Since many of the sequences studied are sites of protein recognition, the observed sequence-structure dependence in DNA probably plays an important role in the process of recognition by proteins and minor groove ligands such as drugs.

NMR spectra of exchangeable protons of pyridoxal phosphate-dependent enzymes.

We have recorded 1H NMR spectra in H2O for exchangeable protons of four pyridoxal phosphate-dependent enzymes: D-serine dehydratase, aspartate aminotransferase, tryptophan: indole-lyase and glutamate decarboxylase. The molecular masses range from 48-250 kDa. In every case there are downfield peaks which are lost when the apoenzyme is formed. In most cases some peaks shift in response to interactions with substrates and inhibitors and with changes in pH. We associate one downfield resonance with the proton on the ring nitrogen of the coenzyme and others with imidazole groups that interact with coenzyme or substrates. The chemical shift for the coenzyme-bound proton differs for free enzyme, substrate Schiff base or quinonoid forms.

Structural characterization of the active site of Brucella abortus Cu-Zn superoxide dismutase: a 15N and 1H NMR investigation.

Prokaryotic Cu-Zn superoxide dismutases (SODs) are rare and poorly characterized compared to their eukaryotic counterparts. To better characterize the structure of the prokaryotic enzyme, an NMR investigation of Brucella abortus Cu-Zn SOD in the reduced form was undertaken. The enzyme studied was a recombinant form, expressed in Escherichia coli. The enzyme initially lacked a full complement of Cu and Zn ion. After demetallation and remetallation with a stoichiometric amount of Cu and Zn ion, the specific activity of the recombinant B. abortus Cu-Zn SOD was comparable to the specific activity of the bovine enzyme. The 15N and 1H resonances of seven active site histidine imidazole rings were assigned using two-dimensional NMR methods. A self-consistent set of nuclear Overhauser effects between imidazole ring protons was observed, which was in agreement with the predictions of a model based on the X-ray crystallographic structure of the oxidized bovine enzyme (Tainer, J.A., Getzoff, E. D., Beem, K. M., Richardson, J.S., & Richardson, D.C. (1982) J. Mol. Biol. 160, 181-217). These observations strongly suggest that the structure of the active site of the prokaryotic enzyme is similar to that of the eukaryotic enzyme. Differences in the observed and predicted nuclear Overhauser effects could be ascribed to differences in the oxidation state of the Cu ion (Cu(I) in the reduced B. abortus enzyme and Cu(II) in the oxidized bovine enzyme), as much as they could to the different origins of the enzymes. The NMR data were also compared to a similar 1H NMR study of the human enzyme (Bertini, I., Capozzi, F., Luchinat, C., Piccioli, M., & Viezzoli, M. S. (1991) Eur. J. Biochem. 197, 691-697). The pattern of nuclear Overhauser effects and the chemical shifts of corresponding resonances were very similar in 1H NMR spectra of the human and B. abortus enzymes. Significant differences in the chemical shifts or exchange behavior of a few resonances indicated differences in the environments of several histidines in the active sites of reduced B. abortus and human Cu-Zn SODs. This is consistent with the presence of a number of insertions and deletions in the loop regions that make up the active site as indicated by amino acid sequence alignment studies. The tautomeric and protonation states of the active site histidines were also determined in this study, and the results were in agreement with previous studies. The resonances of nitrogen atoms coordinated to metal ions were found to fall between those of protonated and unprotonated nitrogens on histidine imidazoles.

Coherence transfer in deoxyribose sugars produced by isotropic mixing: an improved intraresidue assignment strategy for the two-dimensional NMR spectra of DNA.

Two-dimensional isotropic mixing spectroscopy has been used to confirm assignments of the deoxyribose sugar protons in the H NMR spectrum of the DNA oligonucleotides d(CGCGTTTTCGCG) and [d(GCCGTGGCCACGGC)]2. The broad-band decoupling sequence MLEV-16 was applied during the mixing period to induce isotropic coupling within the spin systems, resulting in net transfer of coherence throughout the coupled spin networks. Nearly all 1', 2', 2'', 3', and 4' protons of a given nucleotide could be identified on the basis of through-bond scalar connectivities. In addition, in the hairpin, a number of connectivities to 5'/5'' protons were found. The dependence of cross-peak intensity on the length of radio-frequency irradiation for several different coherence transfer orders is presented, and implications for optimization are discussed.

Nuclear magnetic resonance studies of amino acids and proteins. Deuterium nuclear magnetic resonance relaxation of deuteriomethyl-labeled amino acids in crystals and in Halobacterium halobium and Escherichia coli cell membranes.

We have obtained deuterium (2H) Fourier transform nuclear magnetic resonance (NMR) spectra of zwitterionic L-[beta-2H3]alanine, DL-[gamma-2H6]valine, DL-[beta, gamma-2H4]threonine, L-[delta-2H3]leucine, and L-[alpha, beta, gamma, gamma', delta-2H10]isoleucine in the crystalline solid state and have determined the deuteriomethyl group spin-lattice relaxation rates as a function of temperature. The results yield the Arrhenius activation energies (delta E) for methyl rotation, and through use of a suitable mathematical model, rotational correlation times, tau c. For alanine, valine, threonine, leucine, and isoleucine at 37 degrees C, tau c and delta E values are 780, 100, 40, 38, and 18 ps and 22, 14.0, 17.6, 15.5, and 8.6 kJ, respectively. For L-[beta-2H3]alanine in the zwitterionic lattice, a spin-lattice relaxation time (T1) minimum of 2.1 +/- 0.3 ms is observed (at 0 degree C), in excellent agreement with the 1.92-ms prediction of the mathematical model. Similar tau c and delta E measurements are reported for bacteriorhodopsin in the purple membrane of Halobacterium halobium R1 and for Escherichia coli cell membranes. Overall, our results demonstrate a great similarity between the dynamics in amino acid crystals and in membrane proteins. However, threonine exhibits a nonlinear Arrhenius behavior in bacteriorhodopsin, and in the valine-, leucine-, and isoleucine-labeled membrane samples at higher temperatures (approximately greater than 37 degrees C), there is evidence of an additional slow side-chain motion. The lipid phase state in E. coli does not appear to influence, on the average, the dynamics of the valine side chains. These results indicate that the sensitivity of the deuterium NMR technique is now adequate to study in moderate detail the dynamics of most types of amino acids in a membrane protein and that adequate sensitivity, in some instances, should be available for the study of individual amino acids in suitably labeled membrane proteins.

Effect of citrulline for arginine replacement on the structure and turnover of phosphopeptide substrates of protein phosphatase-1.

Phosphorylated and nonphosphorylated forms of a decapeptide corresponding to residues 9 to 18 of glycogen phosphorylase were compared using two-dimensional nuclear magnetic resonance with assignment of both peptides done by the sequential method. Both forms had little secondary structure, but there was evidence for an interaction between arginine-16 and phosphorylated serine at position 14. A change in the chemical shift for the epsilon-nitrogen hydrogen of arginine in position 16 was observed in the spectrum of the phosphorylated peptide and was not evident in a phosphopeptide having citrulline in place of arginine-16. Hydrolysis catalyzed by protein phosphatase-1 was decreased with the citrulline-containing phosphopeptide compared to the arginine-containing phosphopeptide with effects observed on both kcat and Km of the phosphatase reaction. Alkaline phosphatase hydrolyzed these peptides and a di-citrulline peptide equally well. These results are consistent with arginine being favorable in the recognition of substrates by phosphatase-1, possibly recognition as an arginine-phosphoserine complex. As a model study, arginine and two analogs, citrulline and canavanine, were examined for association with inorganic phosphate by nuclear magnetic resonance spectrometry. 31P-NMR measurements showed that arginine and canavanine caused a shift in the phosphate resonance at 20 degreesC. Citrulline caused no change. Changes in chemical shift were measured over the pH range 5-9 with arginine and canavanine both causing a slight decrease in the apparent pKa of inorganic phosphate (DeltapKa approximately 0.15). NaCl, NH4Cl, and guanidine hydrochloride showed little effect on the resonance signal position of inorganic phosphate at pH 6.5, consistent with selectivity for the guanidino group. Temperature (6 degrees, 20 degrees, and 37 degreesC) caused little change in the effect of arginine, but there was some dependency with canavanine, decreasing with temperature. Citrulline caused no change in the chemical shift of phosphate at any temperature. It was concluded that hydrogen bonded complexes were formed between the dianion of phosphate and the protonated form of arginine or canavanine with a bifurcated structure having preference for the omega-hydrogens.

NMR observation of exchangeable protons of pyridoxal phosphate and histidine residues in cytosolic aspartate aminotransferase.

Observation of the 93-kDa cytosolic aspartate aminotransferase by 500-MHz 1H NMR spectroscopy in H2O has revealed a series of resonances in the 10-18 ppm range arising from exchangeable protons. One of these (peak A) has been assigned to the proton bound to the ring nitrogen of the coenzyme pyridoxal 5'-phosphate. A second (peak B) is assigned to H143 which participates in a chain of hydrogen bonds that includes also the coenzyme-bound proton. There is a mutual nuclear Overhauser effect between these two resonances. Peaks A and B respond to changes in pH and to interaction of the enzyme with coenzyme derivatives and inhibitors. Peak A moves from 15.4 to 17.4 ppm as the pH is lowered, while peak B moves in the opposite direction from 14.7 to 13.7 ppm, both with an apparent pKa of 6.15. This pKa is associated with deprotonation of the imine nitrogen at the Schiff base linkage of the coenzyme with K258 of the enzyme. In spectra of enzyme containing pyridoxamine 5'-phosphate, peak A is observed at 16.5 ppm and peak B is at 13.9 ppm over a broad pH range. Peaks A and B are found at 17.8 and 14.0 ppm, respectively, for the enzyme complex with glutarate. When alpha-methylaspartate is added to the enzyme several new resonances appear in the spectrum, which are attributed to formation of the external aldimine. The position of peak A in spectra of various forms of the enzyme is interpreted to reflect the electronic distribution in the coenzyme ring. Several other peaks in this region of the spectrum also are sensitive to changes in pH or the addition of inhibitors. Some possible assignments of these resonances are discussed.

Dynamics of bases in hydrated [d(CGCGAATTCGCG)]2.

Solid-state 2H NMR spectroscopy has been used to investigate the dynamics of a DNA oligonucleotide with a defined sequence, [d(CGCGAATTCGCG)]2, which contains the EcoRI binding site. Quadrupole echo line shapes and spin-lattice relaxation times were obtained as a function of hydration on two different deuterated samples, both in the form of the Na salt. In one sample, the C8 protons of all purines in the self-complementary dodecamer were exchanged for deuterons. In the other sample, a specifically labeled thymidine (C6 deuterated) was synthetically incorporated at the seventh position (counting 5' to 3') in the sequence. The general trends for both samples were quite similar. At all levels of hydration, the data reveal the presence of a rapid, small-amplitude libration of the bases (tau c less than or equal to 1 ns, 6 degrees-10 degrees amplitude). At the higher hydration levels (80% relative humidity or higher), the results indicate the presence of a much slower motion (tau c approximately 10-100 microseconds), which at 80% relative humidity is of small amplitude (approximately 5 degrees) and at higher hydration levels may be of larger amplitude. There is no evidence for large-amplitude (greater than +/- 10 degrees) motion on a nanosecond or faster time scale under any hydration condition. The 2H NMR results were analyzed with a dynamical model which treats the oligonucleotide as a deformable filament and which can include collective torsional fluctuations. The slow motion observed at high hydration levels is attributed to the uniform twisting mode (of the entire helix).(ABSTRACT TRUNCATED AT 250 WORDS)

Use of 1H-15N heteronuclear multiple-quantum coherence NMR spectroscopy to study the active site of aspartate aminotransferase.

Aspartate aminotransferase from Escherichia coli, an 88 kDa enzyme, was uniformly and selectively enriched with 15N and was studied by heteronuclear multiple-quantum coherence NMR spectroscopy in H2O. Good resolution was obtained for the downfield region (above 9.5 ppm chemical shift in the 1H dimension) for NH protons in the amide, indole, imidazole, and guanidinium group regions and several resonances were tentatively assigned. Two downfield resonances, at 12.6 and 11.36 ppm, appear to belong to oxygen- or sulfur-bound protons. The most downfield amide resonance at 11.78 ppm was assigned to the active site cysteine 192 whose peptide proton is 2.9 A away from the negatively charged carboxyl group of aspartate 199. Large downfield shifts (up to 1.15 ppm) of the indole NH resonance of the active site tryptophan 140 were observed upon binding of dicarboxylic inhibitors to the pyridoxal 5'-phosphate (PLP) form and of inorganic dianions to the pyridoxamine 5'-phosphate (PMP) form of the enzyme. We discuss these striking differences in the light of the available crystallographic data. Active sites of proteins, as well as specific inhibitory molecules, often contain negatively charged groups. These may be able to form hydrogen-bonds to NH groups and to shift the NH resonances downfield into a less crowded and therefore more readily observable region for many large proteins. Our approach, which makes use of both HMQC spectroscopy and NOE observations, should be widely applicable.

First observation of amino acid side chain dynamics in membrane proteins using high field deuterium nuclear magnetic resonance spectroscopy.

We have obtained the first deuterium NMR spectra of an individual membrane protein, bacteriorhodopsin in the purple membrane of Halobacterium halobium R1. Biosynthetic isotopic enrichment with [gamma-2H6]valine and high field Fourier transform operation permitted rapid data acquisition on intact membranes, including measurement of relaxation times. At some temperatures high quality spectra could be obtained in less than 1 s. [U-14C]Valine tracer studies indicate that less than or equal to 2% of valine added to the growth medium is broken down and incorporated into other membrane constituents. The NMR results indicate that the valine side chain is a rather rigid structure. Motion about C alpha-C beta is slow (less than 10(5) s-1) at growth temperature, While motion about C beta-C gamma is as expected fast (much greater than 10(5) s-1) at all accessible temperatures. The activation energy for methyl group rotation from spin-lattice relaxation data between -75 and 53 degrees C is approximately 2.4 kcal/mol, in good agreement with previous 1H NMR studies on solid alkanes. Preliminary data on [gamma-2H6]valine-labeled Acholeplasma laidlawii B (PG9) cell membranes are also presented. Our results strongly suggest that it should now be possible to observe in great detail the motions of any type of amino acid side chain in membrane proteins, including the effects of lipid composition on protein dynamics.