Solution-state structure by NMR of zinc-substituted rubredoxin from the marine hyperthermophilic archaebacterium Pyrococcus furiosus.

The three-dimensional solution-state structure is reported for the zinc-substituted form of rubredoxin (Rd) from the marine hyperthermophilic archaebacterium Pyrococcus furiosus, an organism that grows optimally at 100 degrees C. Structures were generated with DSPACE by a hybrid distance geometry (DG)-based simulated annealing (SA) approach that employed 403 nuclear Overhauser effect (NOE)-derived interproton distance restraints, including 67 interresidue, 124 sequential (i-j = 1), 75 medium-range (i-j = 2-5), and 137 long-range (i-j > 5) restraints. All lower interproton distance bounds were set at the sum of the van Der Waals radii (1.8 A), and upper bounds of 2.7 A, 3.3 A, and 5.0 A were employed to represent qualitatively observed strong, medium, and weak NOE cross peak intensities, respectively. Twenty-three backbone-backbone, six backbone-sulfur (Cys), two backbone-side chain, and two side chain-side chain hydrogen bond restraints were include for structure refinement, yielding a total of 436 nonbonded restraints, which averages to > 16 restraints per residue. A total of 10 structures generated from random atom positions and 30 structures generated by molecular replacement using the backbone coordinates of Clostridium pasteurianum Rd converged to a common conformation, with the average penalty (= sum of the square of the distance bounds violations; +/- standard deviation) of 0.024 +/- 0.003 A2 and a maximum total penalty of 0.035 A2. Superposition of the backbone atoms (C, C alpha, N) of residues A1-L51 for all 40 structures afforded an average pairwise root mean square (rms) deviation value (+/- SD) of 0.42 +/- 0.07 A. Superposition of all heavy atoms for residues A1-L51, including those of structurally undefined external side chains, afforded an average pairwise rms deviation of 0.72 +/- 0.08 A. Qualitative comparison of back-calculated and experimental two-dimensional NOESY spectra indicate that the DG/SA structures are consistent with the experimental spectra. The global folding of P. furiosus Zn(Rd) is remarkably similar to the folding observed by X-ray crystallography for native Rd from the mesophilic organism C. pasteurianum, with the average rms deviation value for backbone atoms of residues A1-L51 of P. furiosus Zn(Rd) superposed with respect to residues K2-V52 of C. pasteurianum Rd of 0.77 +/- 0.06 A. The conformations of aromatic residues that compose the hydrophobic cores of the two proteins are also similar. However, P. furiosus Rd contains several unique structural elements, including at least four additional hydrogen bonds and three potential electrostatic interactions.(ABSTRACT TRUNCATED AT 400 WORDS)

Comparison of the X-ray structure of native rubredoxin from Pyrococcus furiosus with the NMR structure of the zinc-substituted protein.

The three-dimensional X-ray structures of the oxidized and reduced forms of rubredoxin from Pyrococcus furiosus, determined at -161 degrees C, and the NMR structure of the zinc-substituted protein, determined in solution at 45 degrees C, are compared. The NMR and X-ray structures, which were determined independently, are very similar and lead to similar conclusions regarding the interactions that confer hyperthermostability.

Nucleocapsid zinc fingers detected in retroviruses: EXAFS studies of intact viruses and the solution-state structure of the nucleocapsid protein from HIV-1.

All retroviral nucleocapsid (NC) proteins contain one or two copies of an invariant 3Cys-1His array (CCHC = C-X2-C-X4-H-X4-C; C = Cys, H = His, X = variable amino acid) that are essential for RNA genome packaging and infectivity and have been proposed to function as zinc-binding domains. Although the arrays are capable of binding zinc in vitro, the physiological relevance of zinc coordination has not been firmly established. We have obtained zinc-edge extended X-ray absorption fine structure (EXAFS) spectra for intact retroviruses in order to determine if virus-bound zinc, which is present in quantities nearly stoichiometric with the CCHC arrays (Bess, J.W., Jr., Powell, P.J., Issaq, H.J., Schumack, L.J., Grimes, M.K., Henderson, L.E., & Arthur, L.O., 1992, J. Virol. 66, 840-847), exists in a unique coordination environment. The viral EXAFS spectra obtained are remarkably similar to the spectrum of a model CCHC zinc finger peptide with known 3Cys-1His zinc coordination structure. This finding, combined with other biochemical results, indicates that the majority of the viral zinc is coordinated to the NC CCHC arrays in mature retroviruses. Based on these findings, we have extended our NMR studies of the HIV-1 NC protein and have determined its three-dimensional solution-state structure. The CCHC arrays of HIV-1 NC exist as independently folded, noninteracting domains on a flexible polypeptide chain, with conservatively substituted aromatic residues forming hydrophobic patches on the zinc finger surfaces. These residues are essential for RNA genome recognition, and fluorescence measurements indicate that at least one residue (Trp37) participates directly in binding to nucleic acids in vitro. The NC is only the third HIV-1 protein to be structurally characterized, and the combined EXAFS, structural, and nucleic acid-binding results provide a basis for the rational design of new NC-targeted antiviral agents and vaccines for the control of AIDS.

C-terminal retroviral-type zinc finger domain from the HIV-1 nucleocapsid protein is structurally similar to the N-terminal zinc finger domain.

Two-dimensional NMR spectroscopic and computational methods were employed for the structure determination of an 18-residue peptide with the amino acid sequence of the C-terminal retroviral-type (r.t.) zinc finger domain from the nucleocapsid protein (NCP) of HIV-1 [Zn(HIV1-F2)]. Unlike results obtained for the first retroviral-type zinc finger peptide, Zn(HIV1-F1), [Summers et al. (1990) Biochemistry 29, 329], broad signals indicative of conformational lability were observed in the 1H NMR spectrum of Zn-(HIV1-F2) at 25 degrees C. The NMR signals narrowed upon cooling to -2 degrees C, enabling complete 1H NMR signal assignment via standard two-dimensional (2D) NMR methods. Distance restraints obtained from qualitative analysis of 2D nuclear Overhauser effect (NOESY) data were used to generate 30 distance geometry (DG) structures with penalties (penalty = sum of the squared differences between interatomic distances defined in the restraints file and in the DG structures) in the range 0.02-0.03 A2. All structures were qualitatively consistent with the experimental NOESY spectrum based on comparisons with 2D NOESY back-calculated spectra. Superposition of the backbone atoms (C, C alpha, N) for residues C(1)-C(14) gave pairwise RMSD values in the range 0.16-0.75 A. The folding of Zn(HIV1-F2) is very similar to that observed for Zn(HIV1-F1). Small differences observed between the two finger domains are localized to residues between His(9) and Cys(14), with residues M(11)-C(14) forming a 3(10) helical corner.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular structure of charybdotoxin: retraction.

Shortly after our paper of 3 August 1990 on the molecular structure of charybdotoxin (1) was published, two independent determinations of the structure of this molecule appeared (2) that were similar to each other and in strong disagreement with ours. We have obtained new data and find that some spectral features depend on solvent conditions, which explains some differences between our data and those of the other groups. More important, we conclude that we most probably misassigned an important sequence of amino acids, as suggested by Bontems et al.(3). Therefore, we withdraw our previously reported structure (1) and regret any inconvenience it may have caused. We thank F. Toma for sending us a copy of his paper before publication and for discussions.

Molecular structure of charybdotoxin, a pore-directed inhibitor of potassium ion channels.

The three-dimensional structure of charybdotoxin, a high-affinity peptide blocker of several potassium ion channels, was determined by two-dimensional nuclear magnetic resonance (2-D NMR) spectroscopy. Unambiguous NMR assignments of backbone and side chain hydrogens were made for all 37 amino acids. The structure was determined by distance geometry and refined by nuclear Overhauser and exchange spectroscopy back calculation. The peptide is built on a foundation of three antiparallel beta strands to which other parts of the sequence are attached by three disulfide bridges. The overall shape is roughly ellipsoidal, with axes of approximately 2.5 and 1.5 nanometers. Nine of the ten charged groups are located on one side of the ellipsoid, with seven of the eight positive residues lying in a stripe 2.5 nanometers in length. The other side displays three hydrophobic residues projecting prominently into aqueous solution. The structure rationalizes several mechanistic features of charybdotoxin block of the high-conductance Ca2(+)-activated K+ channel.

High-resolution structure of an HIV zinc fingerlike domain via a new NMR-based distance geometry approach.

A new method is described for determining molecular structures from NMR data. The approach utilizes 2D NOESY back-calculations to generate simulated spectra for structures obtained from distance geometry (DG) computations. Comparison of experimental and back-calculated spectra, including analysis of cross-peak buildup and auto-peak decay with increasing mixing time, provides a quantitative measure of the consistence between the experimental data and generated structures and allows for use of tighter interproton distance constraints. For the first time, the "goodness" of the generated structures is evaluated on the basis of their consistence with the actual experimental data rather than on the basis of consistence with other generated structures. This method is applied to the structure determination of an 18-residue peptide with an amino acid sequence comprising the first zinc fingerlike domain from the gag protein p55 of HIV. This is the first structure determination to atomic resolution for a retroviral zinc fingerlike complex. The peptide [Zn(p55F1)] exhibits a novel folding pattern that includes type I and type II NH-S tight turns and is stabilized both by coordination of the three Cys and one His residues to zinc and by extensive internal hydrogen bonding. The backbone folding is significantly different from that of a "classical" DNA-binding zinc finger. Residues C(1)-F(2)-N(3)-C(4)-G(5)-K(6) fold in a manner virtually identical with the folding observed by X-ray crystallography for related residues in the iron domain of rubredoxin; superposition of all main-chain and Cys side-chain atoms of residues C(1)-K(6) of Zn(p55F1) onto residues C(6)-Y(11) and C(39)-V(44) of rubredoxin gives RMSDs of 0.46 and 0.35 A, respectively. The side chains of conservatively substituted Phe and Ile residues implicated in genomic RNA recognition form a hydrophobic patch on the peptide surface.

Solution structure of the EcoRI DNA sequence: refinement of NMR-derived distance geometry structures by NOESY spectrum back-calculations.

The solution structure of the self-complementary DNA duplex [d(CGCGAATTCGCG)]2, which contains the EcoRI restriction site sequence GAATTC at the center, has been studied by two-dimensional nuclear magnetic resonance spectroscopy. Time-dependent nuclear Overhauser effect spectra were used to obtain the initial cross-relaxation rates between 155 pairs of protons. These initial cross-relaxation rates were converted into interproton distances and entered into a distance (bounds) matrix. A distance geometry algorithm (DSPACE) was used to create embedded starting structures and to refine these structures until they showed good agreement with the distance matrix; symmetry constraints were included in the refinement procedure, making the two strands in the refined distance geometry structures virtually identical and significantly improving the agreement with the distance matrix. The NOESY spectrum for one of these distance geometry structures was then calculated from the explicit coordinates by numerically integrating all the z-magnetization transfer pathways among neighboring protons within a specified radius. Distances in this distance geometry structure that did not agree with the experimental NOESY time course were then adjusted accordingly. This process was iterated until a good agreement between calculated and experimental NOESY spectra was reached. The final structure, which generates good agreement with the experimental NOESY spectrum, displays kinks at the C3-G4 base step and at the A6-T7 base step that appear to be similar to those reported for the EcoRI restriction site DNA bound to its endonuclease. The solution structure is not the same as the crystal structure of this DNA duplex.

Three-dimensional solution structure of a DNA duplex containing the BclI restriction sequence: two-dimensional NMR studies, distance geometry calculations, and refinement by back-calculation of the NOESY spectrum.

A three-dimensional solution structure for the self-complementary dodecanucleotide [d-(GCCTGATCAGGC)]2 has been determined by distance geometry with further refinements being performed after back-calculation of the NOESY spectrum. This DNA dodecamer contains the hexamer [d(TGATCA)]2 recognized and cut by the restriction endonuclease BclI, and its structure was determined in hopes of obtaining a better understanding of the sequence-specific interactions which occur between proteins and DNA. Preliminary examination of the structure indicates the structure is underwound with respect to idealized B-form DNA though some of the local structural parameters (glycosyl torsion angle and pseudorotation angle) suggest a B-family type of structure is present. This research demonstrates the requirements (resonance assignments, interproton distance measurements, distance geometry calculations, and NOESY spectra back-calculation) to generate experimentally self-consistent solution structures for short DNA sequences.

Limited sampling of conformational space by the distance geometry algorithm: implications for structures generated from NMR data.

Calculations with a metric matrix distance geometry algorithm were performed that show that the standard implementation of the algorithm generally samples a very limited region of conformational space. This problem is most severe when only a small amount of distance information is used as input for the algorithm. Control calculations were performed on linear peptides, disulfide-linked peptides, and a double-stranded DNA decamer where only distances defining the covalent structures of the molecules (as well as the hydrogen bonds for the base pairs in the DNA) were included as input. Since the distance geometry algorithm is commonly used to generate structures of biopolymers from distance data obtained from NMR experiments, simulations were performed on the small globular protein basic pancreatic trypsin inhibitor (BPTI) that mimic calculations performed with actual NMR data. The results on BPTI and on the control peptides indicate that the standard implementation of the algorithm has two main problems: first, that it generates extended structures; second, that it has a tendency to consistently produce similar structures instead of sampling all structures consistent with the input distance information. These results also show that use of a simple root-mean-square deviation for evaluating the quality of the structures generated from NMR data may not be generally appropriate. The main sources of these problems are identified, and our results indicate that the problems are not a fundamental property of the distance geometry algorithm but arise from the implementations presently used to generate structures from NMR data. Several possible methods for alleviating these problems are discussed.

Determination of DNA structures by NMR and distance geometry techniques: a computer simulation.

Computer simulations have been performed to determine how accurately and precisely structures of DNA oligomers can be generated from distance data obtained from two-dimensional NMR experiments. A hexamer fragment d(CGAATT) of the Dickerson dodecamer [Drew, H.R., Wing, R.M., Takano, T., Broka, C., Tanaha, S., Itakura, K. & Dickerson, R.E. (1981) Proc. Natl. Acad. Sci. USA 78, 2179-2183] was used as the model structure in these simulations. Protons were added to the coordinates of the original x-ray structure, which was then subjected to a regularization procedure to minimize deviations from standard bond lengths and bond angles. The proton-proton distances normally observed in NMR experiments were measured from this regularized target structure and used as input for a distance geometry algorithm. Distance geometry structures were generated from two distance sets, one with essentially exact distances (+/- 0.005 A) and one set with a precision (+/- 0.2 A) that simulates an optimal NMR experiment. The results of these calculations were used to judge how accurately and precisely the following helical parameters could be reproduced from this simulated NMR distance data: helical twist, helical rise, dislocation, roll, tilt, glycosidic angle, delta torsion angle, and pseudorotation angle. These data provide a basis from which to judge the quality of DNA structures produced from real NMR experiments.

Three-dimensional structure of the wild-type lac Pribnow promoter DNA in solution. Two-dimensional nuclear magnetic resonance studies and distance geometry calculations.

The solution structure of a 12 base-pair DNA duplex containing the wt-lac promoter Pribnow sequence TATGTT has been studied by two-dimensional nuclear magnetic resonance spectroscopy. Proton assignments for the 24 sugar and base residues were obtained from two-dimensional correlated nuclear magnetic resonance and two-dimensional nuclear Overhauser effect spectra in both 2H2O and H2O, and by two-dimensional relayed coherence transfer nuclear magnetic resonance spectroscopy experiments. Time-dependent, two-dimensional nuclear Overhauser effect spectra were used to determine the initial cross-relaxation rates between 212 pairs of assigned protons, leading to 212 interproton distances in the double helix (8 to 9 per nucleotide). These distance constraints, and known bond lengths and angles, were entered into a distance matrix. After smoothing the bounds of the distance matrix, 12 trial matrices within the bounds constraints were independently generated and embedded in three-dimensional space using a distance geometry algorithm, to generate 12 trial structures. These trial structures were then refined until they no longer violated the distance matrix. The resulting structures are very similar at the local base-pair and nearest-neighbor base-pair level, but exhibit increasing variation at more distant and global levels. At the nearest-neighbor level, the A to T step and the G to T step within the Pribnow hexamer, as well as the G to T step preceding the hexamer, all exhibit very low screw pitch, i.e. 5(+/- 6) degrees. Conversely, the T to G step in the center of the promoter has a large screw pitch (47(+/- 2) degrees) and the T to G step at the 3' end of the promoter has a very large screw pitch (60(+/- 3) degrees). The limitations of nuclear magnetic resonance spectroscopy distance determination of structure are discussed in terms of resolution and spectral overlap of two-dimensional nuclear Overhauser effect crosspeaks. In the present duplex, the inability to measure several 1'-2' and 1'-2" distances resulted in underdetermination of the precise local sugar conformation for seven of the 24 residues, although the spatial position of all sugars was well defined.

Solution structures of the rabbit neutrophil defensin NP-5.

Solution structures of the rabbit neutrophil defensin NP-5 have been determined by 1H nuclear magnetic resonance (n.m.r.) spectroscopy and distance geometry techniques. This 33 amino acid peptide is part of the oxygen-independent mammalian defense system against microbial infection. The structures were generated from 107 n.m.r. derived inter-residue proton-proton distance constraints. A distance geometry algorithm was then used to determine the range of structures consistent with these distance constraints. These distance geometry calculations employed an improved algorithm that allowed the chirality constraints to be relaxed on prochiral centers when it was not possible to make stereo-specific assignments of protons on these centers. This procedure gave superior results compared with standard distance geometry methods and also produced structures that were more consistent with the original n.m.r. data. Analysis of the NP-5 structures shows that the overall folding of the peptide backbone is well defined by the n.m.r. distance information but that the side-chain group conformations are generally less well defined.

Three-dimensional structure of a DNA hairpin in solution: two-dimensional NMR studies and distance geometry calculations on d(CGCGTTTTCGCG).

The three-dimensional structure of d(CGCGTTTTCGCG) in solution has been determined from proton NMR data by using distance geometry methods. The rate of dipolar cross-relaxation between protons close together in space is used to calculate distances between proton pairs within 5 A of each other; these distances are used as input to a distance geometry algorithm that embeds this distance matrix in three-dimensional space. The resulting refined structures that best agree with the input distances are all very similar to each other and show that the DNA sequence forms a hairpin in solution; the bases of the loop region are stacked, and the stem region forms a right-handed helix. The advantages and limitations of the technique, as well as the computer requirements of the algorithm, are discussed.

Complete assignment of the imino protons of Escherichia coli valine transfer RNA: two-dimensional NMR studies in water.

The imino proton spectrum of Escherichia coli valine tRNA has been studied by two-dimensional nuclear Overhauser effect spectroscopy (NOESY) in H2O solution. The small nuclear Overhauser effects from the imino proton of an internal base pair to the imino protons of each nearest neighbor can be observed as off-diagonal cross-peaks. In this way most of the sequential NOE connectivity trains for all the helices in this molecule can be determined in a single experiment. AU resonances can be distinguished from GC resonances by the AU imino NOE to the aromatic adenine C2-H, thus leading to specific base-pair assignments. In general, the NOESY spectrum alone is not capable of assigning every imino proton resonance even in well-resolved tRNA spectra. Multiple proton peaks exhibit more than two cross-peaks, resulting in ambiguous connectivities, and coupling between protons with similar chemical shifts produces cross-peaks that are incompletely resolved from the diagonal. The sequence of the particular tRNA determines the occurrence of the latter problem, which can often be solved by careful one-dimensional experiments. The complete imino proton assignments of E. coli valine tRNA are presented.

Duplex-hairpin transitions in DNA: NMR studies on CGCGTATACGCG.

Two dimensional NMR methods have been used to assign proton resonances in the high salt (greater than or equal to 100mM Na+), low temperature duplex form of the self-complementary DNA dodecamer d(CGCGTATACGCG). At low salt (less than or equal to 10mM Na+) and higher temperature marked changes in the two-dimensional spectrum, and in the one-dimensional spectrum reported by others, indicate that the molecule converts to an alternate conformation. Using saturation transfer methods, many of the resonances of this new conformation have been assigned, and the kinetics of the interconversion of the two forms has been studied. The linewidth, correlation time, and concentration dependence of the formation of this alternate conformation support the idea that it is a unimolecular hairpin. Observation of chemical shifts and NOEs in the hairpin conformation allow some preliminary structural characterization. Examination of the energetics of the interconversion suggests that the exchange between forms does not proceed through a single stranded intermediate, but rather through another pathway, probably involving a cruciform structure.

Conformation, dynamics, and structural transitions of the TATA box region of self-complementary d[(C-G)n-T-A-T-A-(C-G)n] duplexes in solution.

Structural and kinetic features of the TATA box located in the center of the alternating self-complementary d(C-G-C-G-T-A-T-A-C-G-C-G) duplex (TATA 12-mer) and d(C-G-C-G-C-G-T-A-T-A-C-G-C-G-C-G) duplex (TATA 16-mer) have been probed by high-resolution proton and phosphorus NMR spectroscopy in aqueous solution. The imino exchangeable Watson-Crick protons and the nonexchangeable base protons in the TATA box of the TATA 12-mer and TATA 16-mer duplexes have been assigned from intra and inter base pair nuclear Overhauser effect (NOE) measurements. Imino proton line-width and hydrogen exchange saturation recovery measurements demonstrate that the dA X dT base pairs in the TATA box located in the center of the TATA 12-mer and TATA 16-mer duplexes are kinetically more labile than flanking dG X dC base pairs. The proton and phosphorus NMR parameters of the TATA 12-mer monitor a cooperative premelting transition in the TATA box prior to the onset of the melting transition to unstacked strands. Phosphorus NMR studies have been unable to detect any indication of a right-handed B DNA to a left-handed Z DNA transition for the TATA 12-mer duplex in saturated NaCl solution. By contrast, we do detect the onset of the B to Z transition for the TATA 16-mer in saturated NaCl solution. Proton and phosphorus NMR studies demonstrate formation of a loop conformation with chain reversal at the TATA segment for the TATA 12-mer and TATA 16-mer duplexes on lowering the DNA and counterion concentration. The imino protons (10-11 ppm) and phosphorus resonances (3.5-4.0 ppm; 4.5-5.0 ppm) of the loop segment fall in spectral windows well resolved from the corresponding markers in fully paired segments so tha it should be possible to identify loops in longer DNA helixes. The equilibrium between the loop and fully paired duplex conformations of the TATA 12-mer and TATA 16-mer is shifted toward the latter on addition of moderate salt.

Rotational dynamics of transfer ribonucleic acid: effect of ionic strength and concentration.

We have investigated the influence of ionic strength and nucleic acid concentration on the rotational Brownian motion of Escherichia coli tRNA1Val by studying the decay of the fluorescence polarization anisotropy (FPA) of intercalated ethidium on a nanosecond time scale. The rotational relaxation time tau R remains essentially constant as the ionic strength is varied from 2 to 100 mM at a tRNA concentration of 54 mg/mL. tau R also remains practically unchanged as the tRNA concentration is varied from 0.3 to 54 mg/mL at an ionic strength of 130 mM. Present hydrodynamic theories generally predict a more pronounced concentration dependence for rotational diffusion than we observe. This disagreement may result from a nonrandom distribution of the tRNA molecules in solution due to electrostatic interactions. By combining independent data from time-resolved nuclear Overhauser effect (NOE) cross-relaxation experiments and FPA experiments on the same tRNA, we are able to estimate the interproton spacing for the guanine N1-H and the uracil N3-H of the GU-50 base pair in E. coli tRNA1Val. This distance is 0.272 nm.

Effect of Mg2+ on solution conformation of two different transfer ribonucleic acids.

We have investigated the effect of Mg2+ on the solution conformation of two different tRNAs by studying the decay of the fluorescence polarization anisotropy of intercalated ethidium on a nanosecond time scale. In the presence of endogenous Mg2+, yeast tRNAPhe and Escherichia coli tRNAVal1 exhibit similar behavior; i.e., the fluorescence from the intercalated ethidium decays biexponentially with lifetimes of approximately 25 and approximately 5 ns, and the fluorescence polarization anisotropy decays with a lifetime of approximately 25 ns. However, once Mg2+ is removed from the two tRNAs, their behavior is no longer similar. In the case of yeast tRNAPhe, it appears that titrating with Mg2+ restores the tRNA to the condition that it was in prior to the Mg2+ removal. This is not so for E. coli tRNAVal1, in which case titrating with Mg2+ results in a two-component anisotropy decay with lifetimes of approximately 25 and approximately 6 ns. Rudimentary calculations indicate that the 6-ns component does not result simply from a change in conformation of the tRNA. However, torsional motions in the tRNA facilitated by a torsion "joint" with a rigidity approximately 1/40 that of intact linear phi 29 DNA would yield a decay component on this time scale with about the right amplitude. We are thus left with the possibility that (after initially removing magnesium) titrating tRNAVal1 with Mg2+ leads to increased internal flexibility and a significant amplitude of a deformational relaxation mode. At any rate, there is no question that after removal of Mg2+ tRNAPhe and tRNAVal1 display quite different solution conformation behavior. These findings are in qualitative agreement with recent 500-MHz 1H NMR results from solutions of these two tRNAs.