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.     

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.     

1H-NMR studies of the interaction between a self-complementary deoxyoligonucleotide duplex and indolo[2,3-b]quinoxaline derivatives active against herpes virus

1H NMR has been used to study the interactions of ellipticine and the ellipticine analogues 2-3-dimethyl-6-(2-dimethylaminoethyl)6H-indolo-[2,3-b]quinoxaline and 6-(2-dimethylaminoethyl)6H-indolo-[2,3-b]quinoxaline with the self-complementary decadeoxyribonucleotide d(CGCGATCGCG)2. The Watson-Crick H-bonded imino proton resonances were studied. The drugs were shown to bind to the duplex by intercalation involving slow exchange kinetics for the imino proton resonances on the NMR time scale (500 MHz). Ellipticine and the 2,3-dimethyl analogue were found not to show strong base preferences, while the other analogue was found to have a preferred primary binding site between the A.T base pairs with a probable minor secondary binding site between the A.T and adjacent G.C base pairs. The new drug-shifted imino proton resonances were assigned through saturation transfer experiments. The base-specific interactions were accompanied by drug-induced non-uniform broadening of the resonances (due to intermediate chemical exchange kinetics), in the spectral region of the non-exchangeable aromatic and sugar H1' proton resonances of the oligonucleotide at 25 degrees C.     

Sequential assignment of the 1H and 31P resonances of the double stranded deoxynucleotide d (ATGCAT)2 by 2D-NMR correlation spectroscopy

31p-1H and 1H-1H chemical shift correlation spectroscopy are jointly used for providing a complete assignment of sugar proton (except H5' and H5") and phosphorus resonances in the double stranded oligonucleotide d (ATGCAT)2. In contrast to previous methods the specific assignment of overcrowded H5' H5" proton resonances is not required. Using the H3'-P coupling and also the long range H4'-P coupling, this quite general method can be easily implemented on intermediate field spectrometer. The present results pave the way to the 1H and 31P resonance assignment of longer double-stranded oligonucleotides.     

Two-dimensional proton nuclear magnetic resonance investigation of the synthetic deoxyribonucleic acid decamer d(ATATCGATAT)2

In this study two-dimensional NMR techniques (COSY and NOESY) have been used in conjunction with one-dimensional NMR results to complete the assignment of the proton NMR spectrum of the double-stranded DNA decamer, d(ATATCGATAT)2, and to obtain qualitative information about numerous interproton distances in this molecule and some limited information about conformational dynamics. COSY and NOESY measurements have been combined to systematically assign many of the resonances from the H1' and H2',2" sugar protons to specific nucleotides in the double helix. This method relies on the fact that sugar protons within a specific nucleotide are scalar coupled and that base protons (AH8, GH8, TH6, and CH6) in right-handed helices can interact simultaneously with their own H2',2" sugar protons and those of the adjacent (5'-3') nucleotide attached to its 5' side (i.e., XpA not ApX). A COSY experiment is used to identify sugar resonances within a residue whereas the NOESY experiment allows the neighboring sugar to be connected (linked). The CH5 and CH6 resonances in the spectrum can immediately be identified by the COSY experiment. The methyl protons of thymine residues exhibit strong through-space interbase interactions both with their own TH6 proton and with AH8 proton on the adjacent (5'-3') adenine residue. These interactions are used both to make assignments of the spectra and to establish that the thymine methyl groups are in close proximity to the AH8 protons of adjacent adenine residues [Feigon, J., Wright, J. M., Leupin, W., Denny, W. A., & Kearns, D. R. (1982) J. Am. Chem. Soc. 104, 5540].(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of N6-methylation of residue A(5) on the conformational behaviour of d(C-C-G-A-A-T-T-C-G-G) in solution studied by 1H-NMR spectroscopy. 1. The duplex form

One- and two-dimensional NMR studies at 300 MHz and 500 MHz were carried out on the two oligonucleotides d(C-C-G-A-A-T-T-C-G-G) and d(C-C-G-A-m6A-T-T-C-G-G) in aqueous solution. NMR spectra were observed at 10 mM sample concentration over the temperature range 273-368 K. Assignments are given of the base, H1', H2', H2", H3' and of some H4' resonances, based upon a combination of two-dimensional correlation spectra (COSY) and two-dimensional nuclear Overhauser effect spectra (NOESY); imino-proton resonances were assigned with the aid of a two-dimensional NOE experiment. Chemical shift vs temperature profiles were constructed in order to gain insight into the influence of N6-methylation of residue A(5) on the temperature-dependent conformational behaviour of the decamer and to determine thermodynamic parameters for the duplex-to-coil transition. The NOESY spectra, the imino-proton spectra and the shift profiles of the two compounds, under conditions where each forms a B-DNA-type duplex, are very similar. This is taken to indicate that the influence of N6-methylation of residue A(5) on the local structure of the duplex must be small. However, the temperature dependence of the (non-)exchangeable proton resonances of the two compounds reveals that methylation slows down the duplex-single-strand exchange. Furthermore, a thermodynamic analysis of the two compounds indicates that N6-methylation slightly decreases the stability of the duplex relative to the monomeric forms (Tm is reduced from 332 K down to 325 K at 10 mM sample concentration). Proton-proton couplings were obtained by means of one-dimensional and two-dimensional NMR experiments and were used in a conformational analysis of the sugar ring of each residue of the two compounds in the duplex form. The analysis indicated that all sugar rings display conformational flexibility in the intact duplex: population S-type sugar conformation ranges from 70% to 100%. A more refined analysis of the sugar rings of the parent compound revealed a sequence-dependent variation of the sugar geometry. This variation does not follow well the trend predicted by the Calladine/Dickerson sigma 3-sum rule [Dickerson, R. E. (1983) J. Mol. Biol. 166, 419-441; Calladine, C. R. (1982) J. Mol. Biol. 161, 343-352]; moreover the actual variations appear to be smaller in solution than those expected on the basis of known X-ray structures.     

The rotating frame nuclear Overhauser effect spectroscopy experiment as an aid to determining solution structures of DNA oligomers

Important intrinsic characteristics of the rotating frame nuclear Overhauser effect spectroscopy (ROESY) experiment were found to be advantageous in DNA solution structure determination. In a ROESY experiment, the different mechanisms of relaxation result in different signs of cross peaks, enabling a clear distinction between H2' resonances and H2" resonances of the DNA sugar backbone. This method is of particular importance in crowded spectra, for purine resonances whose H2', H2" protons typically resonate closely, as well as in conditions where line broadening makes coupling constants in a correlated spectroscopy experiment impossible to determine. By observing the signs of cross peaks in the base proton to H2', H2" sugar proton region, the ROESY spectrum can be used to distinguish A-form, B-form, and Z-form DNA.     

Polarity of annealing and structural analysis of the RNase H resistant alpha-5'-d[TACACA].beta-5'-r[AUGUGU] hybrid determined by high-field 1H, 13C, and 31P NMR analysis.

The novel hybrid duplex alpha-5'-d[TACACA]-3'.beta-5'-r[AUGUGU]-3' was analyzed extensively by 1D and 2D NMR methods. Two forms of the duplex exist in about an 80:20 ratio. Analysis of the exchangeable imino protons of the major component revealed that three AU and one AT base pair are present in addition to two GC base pairs, confirming that the duplex anneals in parallel orientation. The presence of the AT base pair, which can only be accounted for by a parallel duplex, was confirmed by a selective INEPT experiment, which correlated the thymidine imino proton to its C5 carbon. The lesser antiparallel form could be detected by exchangeable and nonexchangeable proton resonances in both strands. An exchange peak was observed in the NOESY spectrum for the thymidine methyl group resonance in both the predominant and lesser conformations, indicating the lifetime of the individual structures was on the millisecond time scale. The nonexchangeable protons of the predominant duplex were assigned by standard methods. The sugar pucker of the ribonucleosides was determined to be of the "S" type by a pseudorotation analysis according to Altona, with the J-couplings measured from the multiplet components of the phase-sensitive COSY experiment. The NOE pattern observed for the alpha-deoxynucleosides also suggested an S-type sugar pucker. The adoption of an S-type sugar pucker for both strands indicates that, in contrast to RNA.DNA duplexes formed exclusively from beta-nucleotides, the alpha-DNA.beta-RNA duplex may form a B-type helix. The 31P resonances of the alpha and beta strands have very different chemical shifts in the hybrid duplex and the difference persists above the helix melting temperature, indicating an intrinsic difference in 31P chemical shift for nucleotides differing only in the configuration about the glycosidic bond.     

Assignment of phosphorus-31 and nonexchangeable proton resonances in a symmetrical 14 base pair lac pseudooperator DNA fragment

The 31P chemical shifts of all 13 phosphates and the chemical shifts of nearly all of the non-exchangeable protons of a symmetrical 14 base pair lac pseudooperator DNA fragment have been assigned by regiospecific labeling with oxygen-17 and two-dimensional NMR techniques. At 22 degrees C, 8 of the 13 phosphorus resonances can distinctly be resolved while the remaining 5 resonances occur in two separate overlapping regions. The 31P chemical shifts of this particular 14 base pair oligonucleotide do not follow the general observation that the more internal the phosphate is located within the oligonucleotide sequence the more upfield the 31P resonance occurs, as shown from other 31P assignment studies. Failure of this general rule is believed to be a result of helical distortions that occur along the oligonucleotide double helix, on the basis of the analysis of Callidine [Callidine, C.R. (1982) J. Mol. Biol. 161, 343-352]. Notable exceptions to the phosphate position relationship are 5'-Py-Pu-3' dinucleotide sequences, which resonate at a lower field strength than expected in agreement with similar results as reported by Ott and Eckstein [Ott, J., & Eckstein, F. (1985) Biochemistry 24, 253]. A reasonable correlation exists between 31P chemical shifts values of the 14-mer and the helical twist sum function of Calladine. The most unusual 31P resonance occurs most upfield in the 31P spectrum, which has been assigned to the second phosphate position (5'-GpT-3') from the 5' end. This unusual chemical shift may be the result of the predicted large helical twist angle that occurs at this position in the 14-mer sequence. Further, it is believed that the large helical twist represents a unique structural feature responsible for optimum binding contact between lac repressor protein and this 14-mer lac pseudooperator segment. Assignments of proton resonances were made from two-dimensional 1H-1H nuclear Overhauser effect (NOESY) connectivities in a sequential manner applicable to right-handed B-DNA, in conjunction with two-dimensional homonuclear and heteronuclear J-correlated spectroscopies (1H-1H COSY and 31P-1H HETCOR). Most nonexchangeable base proton and deoxyribose proton (except for some unresolved H4', H5', and H5" protons) resonances were assigned.     

Conformational analysis of the deoxyribofuranose ring in DNA by means of sums of proton-proton coupling constants: a graphical method

A graphical method is presented for the conformational analysis of the sugar ring in DNA fragments by means of proton-proton couplings. The coupling data required for this analysis consist of sums of couplings, which are referred to as sigma 1' (= J1'2' + J1'2'), sigma 2' (= J1'2' + J2'3' + J2'2'), sigma 2' (= J1'2' + J2'3' + J2'2') and sigma 3' (= J2'3' + J2'3' + J3'4'). These sums of couplings correspond to the distance between the outer peaks of the H1', H2', H2' and H3' [31P] resonances, respectively, (except for sigma 2' and sigma 2' in the case of a small chemical shift difference between the H2' and H2' resonances) and can often be obtained from 1H-NMR spectra via first-order measurement, obviating the necessity of a computer-assisted simulation of the fine structure of these resonances. Two different types of graphs for the interpretation of the coupling data are discussed: the first type of graph serves to probe as to whether or not the sugar ring occurs as a single conformer, and if so to analyze the coupling data in terms of the geometry of this sugar ring. In cases where the sugar ring does not occur as a single conformer, but as a blend of N- and S-type sugar puckers, the second type of graph is used to analyze the coupling data in terms of the geometry and population of the most abundant form. It is shown that the latter type of analysis can be carried out on the basis of experimental values for merely sigma 1',sigma 2' and sigma 2', without any assumptions or restrictions concerning a relation between the geometry of the N- and S-type conformer. In addition, the question is discussed as to how insight can be gained into the conformational purity of the sugar ring from the observed fine structure of the H1' resonance. Finally, a comparison is made between experimental coupling data reported for single-stranded and duplex DNA fragments and covalent RNA-DNA hybrids on the one hand and the predicted couplings and sums of couplings presented in this paper on the other hand.     

Sequence-dependent recognition of DNA duplexes: netropsin complexation to the TATA site of the d(G-G-T-A-T-A-C-C) duplex in aqueous solution

We have recorded one-dimensional exchangeable proton and two-dimensional nonexchangeable proton nmr spectra on the complex of netropsin with the self-complementary d(G-G-T-A-T-A-C-C) duplex in aqueous solution between 25° and 35°C. The antibiotic amide, pyrrole, and methylene protons, and the nucleic acid base and sugar H1′, H2′, H2″, and H3′ protons, have been assigned from an analysis of the two-dimensional nuclear Overhauser effect (NOESY) spectra of the complex. We observe intermolecular NOEs between the antibiotic concave face amide, pyrrole, and CH₂ resonances, and the adenosine H2 and sugar H1′ protons of base-pairs T3·A6 and A4·T5 in the central TATA core of the d(G₁-G₂-T₃-A₄-T₅-A₆-C₇-C₈) duplex. We present a molecular model outlining these seven antibiotic-DNA contacts for the complex in solution. The observed line-broadening of several base and sugar protons at the TATA minor groove netropsin binding site in the complex at 35°C are interpreted in terms of intermediate exchange between two orientations of bound netropsin on the duplex.     

NMR studies of echinomycin bisintercalation complexes with d(A1-C2-G3-T4) and d(T1-C2-G3-A4) duplexes in aqueous solution: sequence-dependent formation of Hoogsteen A1.T4 and Watson--Crick T1.A4 base pairs flanking the bisintercalation site

We report on two-dimensional proton NMR studies of echinomycin complexes with the self-complementary d(A1-C2-G3-T4) and d(T1-C2-G3-A4) duplexes in aqueous solution. The exchangeable and nonexchangeable antibiotic and nucleic acid protons in the 1 echinomycin per tetranucleotide duplex complexes have been assigned from analyses of scalar coupling and distance connectivities in two-dimensional data sets recorded in H2O and D2O solution. An analysis of the intermolecular NOE patterns for both complexes combined with large upfield imino proton and large downfield phosphorus complexation chemical shift changes demonstrates that the two quinoxaline chromophores of echinomycin bisintercalate into the minor groove surrounding the dC-dG step of each tetranucleotide duplex. Further, the quinoxaline rings selectively stack between A1 and C2 bases in the d(ACGT) complex and between T1 and C2 bases in the d(TCGA) complex. The intermolecular NOE patterns and the base and sugar proton chemical shifts for residues C2 and G3 are virtually identical for the d(ACGT) and d(TCGA) complexes. A change in sugar pucker from the C2'-endo range to the C3'-endo range is detected at C2 on formation of the d(ACGT) and d(TCGA) complexes. In addition, the sugar ring protons of C2 exhibit upfield shifts and a large 1 ppm separation between the H2' and H2" protons for both complexes. The L-Ala amide protons undergo large downfield complexation shifts consistent with their participation in intermolecular hydrogen bonds for both tetranucleotide complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proton nuclear magnetic resonance investigation of the conformation and dynamics in the synthetic deoxyribonucleic acid decamers d(ATATCGATAT) and d(ATATGCATAT)

A variety of one-dimensional proton NMR methods have been used to investigate the properties of two synthetic DNA decamers, d(ATATCGATAT) and d(ATATGCATAT). These results, in conjunction with the results of two-dimensional NMR experiments, permit complete assignment of the base proton resonances. Low-field resonances were assigned by sequential "melting" of the A . T base pairs and by comparison of the spectra of the two decamers. Below 20 degree C spin-lattice relaxation is dominated by through-space dipolar interactions. A substantial isotope effect on the G imino proton relaxation is observed in 75% D2O, confirming the importance of the exchangeable amino protons in the relaxation process. A somewhat smaller isotope effect is observed on the T imino proton relaxation. At elevated temperatures spin-lattice relaxation of the imino protons is due to proton exchange with solvent. Apparent activation energies for exchange vary from 36 kcal/base pair for base pairs (3,8) to 64 kcal/mol for the most interior base pairs (5,6), indicating that disruption of part, or all, of the double helix contributes significantly to the exchange of the imino protons in these decamers. By contrast, single base pair opening events are the major low-temperature pathways for exchange from A X T and G X C base pairs in the more stable higher molecular weight DNA examined in other studies. The temperature dependence of the chemical shifts and line widths of certain aromatic resonances indicates that the interconversion between the helix and coil states is not in fast exchange below the melting temperature, Tm. Within experimental error, no differential melting of base pairs was found in either molecule, and both exhibited melting points Tm = 50-52 degrees C. Spin-spin and spin-lattice relaxation rates of the nonexchangeable protons (TH6, AH8, and AH2) are consistent with values calculated by using an isotropic rotor model with a rotational correlation time of 6 ns and interproton distances appropriate for B-family DNA. The faster decay of AH8 compared with GH8 is attributed to an interaction between the thymine methyl protons and the AH8 protons in adjacent adenines (5'ApT3'). The base protons (AH8, GH8, and TH6) appear to be located close (1.9-2.3 A) to sugar H2',2" protons.(ABSTRACT TRUNCATED AT 400 WORDS)     

Study of structure, base-pair opening kinetics and proton exchange mechanism of the d-(AATTGCAATT) self-complementary oligodeoxynucleotide in solution.

Using proton magnetic resonance, we have investigated the structure and the base-pair opening kinetics of the d-(AATTGCAATT) self-complementary duplex. All the non-exchangeable (except H5',5") and most exchangeable proton resonances have been assigned. The structure belongs to the B family. Imino proton exchange, measured by line broadening, longitudinal relaxation and magnetization transfer from water, is catalyzed by proton acceptors. The base-pair lifetimes, obtained by extrapolation of the exchange times to infinite concentration of ammonia are 2 and 3 milliseconds for internal A.Ts and 18 ms for G.C at 15 degrees C. In the absence of added catalysts, the imino proton of the first A.T base pair exchanges faster than that of the unpaired thymidine of the duplex formed by the sequence d-(AATTGCAATTT). This gives strong evidence for intrinsic exchange catalysis. The exchange of adenine amino protons from the closed state has been observed. Hence amino proton exchange is ill-suited for the investigation of base-pair opening kinetics.     

The structure of the double-stranded RNA pentamer 5'(CACAG) . 5'(CUGUG) determined by nuclear Overhauser enhancement measurements: interproton distance determination and structure refinement on the basis of X-ray coordinates

The structure in solution of the duplex RNA pentamer 5'(CACAG) . 5'(CUGUG), comprising the stem of the T psi C loop of yeast tRNAPhe, has been investigated by means of one- and two-dimensional nuclear Overhauser enhancement measurements. All non-exchangeable base and sugar proton resonances with the exception of the H5'/H5" sugar resonances are assigned in a sequential manner. From the relative intensities of the cross-peaks obtained in the pure-phase absorption two-dimensional nuclear Overhauser enhancement spectra at several mixing times, it is deduced that the RNA pentamer adopts an A-type conformation in solution. Cross-relaxation rates and interproton distances are determined from the time dependence of the nuclear Overhauser effects, principally by one-dimensional measurements. The structure of the RNA pentamer is then refined by restrained least-squares minimization on the basis of both distance and planarity restraints using fibre diffraction data as an initial model. The refined structure of the RNA pentamer is of the A type but exhibits local structural variations in glycosidic bond and backbone torsion angles as well as in propeller twist, base roll and base tilt angles.     

Resonance assignments of non-exchangeable protons in B type DNA oligomers, an overview.

The chemical shifts of 1H resonances of non exchangeable protons (except H5', H5" and adenine H2) of over six hundred nucleotides have been collected. The influence which the base of the nucleotide itself as well as the bases on its 5' and 3' side exert on the chemical shifts of the various resonances has been investigated. Most of the resonances appear to be predominantly influenced by only one base. For H2', H2", H3', H4' and H6/H8 this is the base of the central nucleotide, for H5(C) and CH3(T) it is the one on the 5' side and for H1' it is the one on the 3' side. Chemical shift distribution profiles are presented which allow an estimation of the probability of finding a particular resonance at a particular position in the spectrum.     

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

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

Characterization of the low-field proton magnetic resonance spectrum of plasminogen kringle 4 via selective Overhauser experiments in 1H2O

The low-field 1H NMR spectrum of the kringle 4 domain of human plasminogen has been investigated at 300 and 600 MHz for the protein dissolved in 1H2O. The spectrum exhibits six well-resolved resonances, spanning the 9.8 approximately less than delta approximately less than 13 ppm chemical shift range, which arise from exchange-labile H atoms. The acid-base response of the six resonances was monitored in order to characterize the signals in terms of their pH titration profiles. The sensitivity of the low-field resonances to kringle binding the antifibrinolytic ligands N alpha-acetyl-L-lysine and p-benzylaminesulfonic acid was also investigated. The lowest field resonance, at 12.6 ppm, is a doublet of J approximately 7.9 Hz, a splitting that is unprecedented for His or Trp ring NH signals. Selective Overhauser experiments centered on the exchangeable proton transitions identify four of the other resonances as stemming from the His31, His33, Trp I, and Trp II side-chain NH groups, where the latter two are, as yet, not definitely assigned to the specific residues, Trp25 and Trp62. The relative narrowness of the His imidazole NH signals indicates that the two rings are sterically shielded from direct water accessibility. In particular, the His33 NH site appears to be the most protected. The Overhauser evidence conclusively shows that the two identified exchangeable His ring proton signals arise from imidazole NH3 sites rather than from the NH1 tautomers. Similarly, these experiments lead to an unambigous characterization of the corresponding Trp aromatic CH spin systems.(ABSTRACT TRUNCATED AT 250 WORDS)     

The solution structure of a RNA pentadecamer comprising the anticodon loop and stem of yeast tRNAPhe. A 500 MHz 1H-n.m.r. study.

A 500 MHz 1H-n.m.r. study on the semi-synthetic RNA pentadecamer 5'-r(C-A-G-A-Cm-U-Gm-A-A-Y-A-psi-m5C-U-G) comprising the anticodon loop and stem (residues 28-42) of yeast tRNAPhe is presented. By using pre-steady-state nuclear-Overhauser-effect measurements all exchangeable and non-exchangeable base proton resonances, all H1' ribose resonances and all methyl proton resonances are assigned and over 70 intra- and inter-nucleotide interproton distances determined. From the distance data the solution structure of the pentadecamer is solved by model-building. It is shown that the pentadecamer adopts a hairpin-loop structure in solution with the loop in a 3'-stacked conformation. This structure is both qualitatively and quantitatively remarkably similar to that of the anticodon loop and stem found in the crystal structures of tRNAPhe with an overall root-mean-square difference of 0.12 nm between the interproton distances determined by n.m.r. and X-ray crystallography. The hairpin-loop solution structure of the pentadecamer is very stable with a 'melting' temperature of 53 degrees C in 500 mM-KCl, and the structural features responsible for this high stability are discussed. Interaction of the pentadecamer with the ribotrinucleoside diphosphate UpUpC, one of the codons for the amino acid phenylalanine, results only in minor perturbations in the structure of the pentadecamer, and the 3'-stacked conformation of the loop is preserved. The stability of the pentadecamer-UpUpC complex (K approximately 2.5 X 10(4) M-1 at 0 degrees C) is approximately an order of magnitude greater than that of the tRNAPhe-UpUpC complex.     

Properties of pseudouridine N1 imino protons located in the major groove of an A-form RNA duplex.

The exchangeable N1 imino protons of two pseudouridine (psi) bases located at adjacent internal positions within an undecamer RNA duplex (5'AUAC psi psi ACCUG/3'UAUGAAUGGUC) can report on the environment of the major groove of an A-form double-stranded nucleic acid. The psi N1 imino protons of these residues (which are not involved in interstrand Watson-Crick hydrogen bonding) are protected from chemical exchange with the solvent water and thus are observable in the proton NMR spectrum in H2O (1). These protons will exchange readily at increased pH values or upon thermal denaturation of the duplex. The longitudinal (T1) relaxation times of the psi N1 imino protons in 100 mM NaCl or in 10 mM MgCl2 and 100 mM NaCl are approximately two-fold faster than those of the psi N3 imino protons which are involved in Watson-Crick base pairing. With the addition of spermidine, the psi N1 imino protons become readily exchangeable at a temperature some 20 degrees C below the melting temperature of the duplex.