Nuclear magnetic resonance signal assignments of purified [13C]methyl-enriched yeast phenylalanine transfer ribonucleic acid

Yeast tRNA Phe, enriched in carbon-13 specifically at the naturally occurring methyl groups, has been produced through biosynthesis, then purified, and analyzed. Transfer RNA Phe was purified from the [13C]methyl-enriched, unfractionated tRNA that had been extracted from a methionine auxotroph of Saccharomyces cerevisiae [Agris, P. F., Kovacs, S. A. H., Smith, C., Kopper, R. H., & Schmidt, P. G. (1983) Biochemistry 22, 1402-1408]. The yeast had been grown in minimal medium supplemented with [13C]methylmethionine. Transfer RNA Phe purity and the full extent of nucleoside modification were confirmed by high-performance liquid chromatography of constituent nucleosides with simultaneous UV spectral identification and quantitation. Mass spectometry of [13C]methyl-enriched nucleosides and NMR of the tRNA indicated an enrichment of at least 70 atom %. Twelve resolved and prominent carbon-13 NMR signals from the tRNA were seen between 10 and 60 ppm. These have been assigned to 13 of the 14 naturally occurring methyl groups. However, the partially resolved signals assigned to the two 5-methylcytidines could not be assigned to their specific nucleoside positions of either 40 or 49 in the molecule. In addition, the partially resolved signals of the two methyl esters of wybutosine could not be distinguished. The methyl group found not to be enriched with 13C is bound to the ring carbon in the hypermodified nucleoside wybutosine (Y). A 13th enriched signal downfield (120.9 ppm) has been assigned to one of the two carbons added to guanosine to form the third ring in the biosynthesis of Y. The 13C enrichment of this ring carbon demonstrates its origin from the methionine methyl group.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of 5-methylcytidine in the anticodon arm of yeast tRNA(Phe): site-specific Mg2+ binding and coupled conformational transition in DNA analogs.

The tDNA(Phe)AC, d(CCAGACTGAAGAU13m5C14U15GG), with a DNA sequence similar to that of the anticodon stem and loop of yeast tRNA(Phe), forms a stem and loop structure and has an Mg(2+)-induced structural transition that was not exhibited by an unmodified tDNA(Phe)AC d(T13C14T15) [Guenther, R. H., Hardin, C. C., Sierzputowska-Gracz, H., Dao, V., & Agris, P. F. (1992) Biochemistry (preceding paper in this issue)]. Three tDNA(Phe)AC molecules having m5C14, tDNA(Phe)AC d(U13m5C14U15), d(U13m5C14T15), and d(T13,5C14U15), also exhibited Mg(2+)-induced structural transitions and biphasic thermal transitions (Tm approximately 23.5 and 52 degrees C), as monitored by CD and UV spectroscopy. Three other tDNA(Phe)AC, d(T13C14T15), d(U13C14U15), and d(A7;U13m5C14U15) in which T7 was replaced with an A, thereby negating the T7.A10 base pair across the anticodon loop, had no Mg(2+)-induced structural transitions and only monophasic thermal transitions (Tm of approximately 52 degrees C). The tDNA(Phe)AC d(U13m5C14U15) had a single, strong Mg2+ binding site with a Kd of 1.09 x 10(-6) M and a delta G of -7.75 kcal/mol associated with the Mg(2+)-induced structural transition. In thermal denaturation of tDNA(Phe)AC d(U13m5C14U15), the 1H NMR signal assigned to the imino proton of the A5.dU13 base pair at the bottom of the anticodon stem could no longer be detected at a temperature corresponding to that of the loss of the Mg(2+)-induced conformation from the CD spectrum. Therefore, we place the magnesium in the upper part of the tDNA hairpin loop near the A5.dU13 base pair, a location similar to that in the X-ray crystal structure of native, yeast tRNA(Phe).(ABSTRACT TRUNCATED AT 250 WORDS)     

Local structural features around the C-terminal segment of Streptomyces subtilisin inhibitor studied by carbonyl carbon nuclear magnetic resonances three phenylalanyl residues

The carbonyl carbon NMR signals of the Phe residues in Streptomyces subtilisin inhibitor (SSI) were selectively observed for [F]SSI, in which all phenylalanines were uniformly labeled with [1-13C]Phe. The three enhanced resonances in the spectrum of [F]SSI were unambiguously assigned to the specific sites in the amino acid sequence by means of 15N,13C double-labeling techniques. Namely, the resonances at 174.9 and 172.6 ppm (in D2O, pH 7.3, 50 degrees C) showed the satellite peaks due to 13C-15N spin coupling in the spectra of [F,GS]SSI and [F,A]SSI, in which Ser/Gly and Ala residues were labeled with [15N]Gly/Ser and [15N]Ala, respectively, together with [1-13C]Phe. The carbonyl groups of Phe-97 and Phe-111 are involved in peptide bonds with the amino nitrogens of Ser-98 and Ala-112, respectively. These results clearly indicate that the signals at 174.5 and 172.6 ppm are due to Phe-97 and Phe-111, respectively. The signal at the lowest field (177.1 ppm) was thus assigned to the carboxyl carbon of the C-terminal Phe-113. The lifetimes of the amide hydrogens of the three Phe residues and their C-terminal-side neighbors (Ser-98 and Ala-112) were investigated by using the effect of deuterium-hydrogen exchange of amide on the line shapes (DEALS) for the Phe carbonyl carbon resonances. In this method, the NMR spectra of [F]SSI dissolved in 50% D2O (pH 7.3) were measured at various temperatures, and the line shape changes caused by deuteriation isotope shifts were analyzed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Three-dimensional 15N-1H-1H and 15N-13C-1H nuclear-magnetic resonance studies of HPr a central component of the phosphoenolpyruvate-dependent phosphotransferase system from Escherichia coli. Assignment of backbone resonances.

We have performed three-dimensional NMR studies on a central component of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli, denoted as HPr. The protein was uniformly enriched with 15N and 13C to overcome spectral overlap. Complete assignments were obtained for the backbone 1H, 15N and 13C resonances, using three-dimensional heteronuclear 1H NOE 1H-15N multiple-quantum coherence spectroscopy (3D-NOESY-HMQC) and three-dimensional heteronuclear total correlation 1H-15N multiple-quantum coherence spectroscopy (3D-TOCSY-HMQC) experiments on 15N-enriched HPr and an additional three-dimensional triple-resonance 1HN-15N-13C alpha correlation spectroscopy (HNCA) experiment on 13C, 15N-enriched HPr. Many of the sequential backbone 1H assignments, as derived from two-dimensional NMR studies [Klevit, R.E., Drobny, G.P. & Waygood, E.B. (1986) Biochemistry 25, 7760-7769], were corrected. Almost all discrepancies are in the helical regions, leaving the published antiparallel beta-sheet topology almost completely intact.     

Solution studies of staphylococcal nuclease H124L. 2. 1H, 13C, and 15N chemical shift assignments for the unligated enzyme and analysis of chemical shift changes that accompany formation of the nuclease-thymidine 3',5'-bisphosphate-calcium ternary complex.

Accurate 1H, 15N, and 13C chemical shift assignments were determined for staphylococcal nuclease H124L (in the absence of inhibitor or activator ion). Backbone 1H and 15N assignments, obtained by analysis of three-dimensional 1H-15N HMQC-NOESY data [Wang, J., Mooberry, E.S., Walkenhorst, W.F., & Markley, J. L. (1992) Biochemistry (preceding paper in this issue)], were refined and extended by a combination of homo- and heteronuclear two-dimensional NMR experiments. Staphylococcal nuclease H124L samples used in the homonuclear 1H NMR studies were at natural isotopic abundance or labeled randomly with 2H (to an isotope level of 50%); nuclease H124L samples used for heteronuclear NMR experiments were labeled uniformly with 15N (to an isotope level greater than 95%) or uniformly with 13C (to an isotope level of 26%). Additional nuclease H124L samples were labeled selectively by incorporating single 15N- or 13C-labeled amino acids. The chemical shifts of uncomplexed enzyme were then compared with those determined previously for the nuclease H124L.pdTp.Ca2+ ternary complex [Wang, J., LeMaster, D. M., & Markley, J.L. (1990) Biochemistry 29, 88-101; Wang, J., Hinck, A.P., Loh, S. N., & Markley, J.L. (1990) Biochemistry 29, 102-113; Wang, J., Hinck, A.P., Loh, S.N., & Markley, J.L. (1990) Biochemistry 29, 4242-4253]. The results reveal that the binding of pdTp and Ca2+ induces large shifts in the resonances of several amino acid segments. These chemical shift changes are interpreted in terms of changes in backbone torsion angles that accompany the binding of pdTp and Ca2+; changes at the binding site appear to be transmitted to other regions of the molecule through networks of hydrogen bonds.     

Structure of transfer RNA by carbon NMR: resolution of single carbon resonances from 13C-enriched, purified species

Methyl carbon-13 NMR spectra of purified tRNA species are presented for the first time. In addition, these spectra of tRNA species specific for phenylalanine, tyrosine, and cysteine exhibited the first resolution of single methyl carbon resonances. Carbon-13 enriched methyl groups of ribothymidine (T) and 7-methylguanosine (m7G) and the methylthio group of 2-methylthio-N6-(delta2-isopentenyl) adenosine (ms2i6A) were resolved. The T methyl signal of tRNAPhe shifted from 12.3 ppm at 45 degrees in the absence of added Mg2+ to 11.1 ppm at 30 degrees in the presence of 10mM MgCl2. The same change in conditions led to a 0.4 ppm shift of the m7G methyl signal in the opposite direction. The relative ease in obtainment of single carbon resonances of purified tRNA species, and display of the sensitivity of their chemical shifts to changes in local structure, are requisite criteria for 13C-NMR to be a useful technique in probing tRNA conformation and its changes during interaction with proteins and other nucleic acids.     

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.     

Assignment of the aliphatic 1H and 13C resonances of the Bacillus subtilis glucose permease IIA domain using double- and triple-resonance heteronuclear three-dimensional NMR spectroscopy.

Nearly complete assignment of the aliphatic 1H and 13C resonances of the IIAglc domain of Bacillus subtilis has been achieved using a combination of double- and triple-resonance three-dimensional (3D) NMR experiments. A constant-time 3D triple-resonance HCA(CO)N experiment, which correlates the 1H alpha and 13C alpha chemical shifts of one residue with the amide 15N chemical shift of the following residue, was used to obtain sequence-specific assignments of the 13C alpha resonances. The 1H alpha and amide 15N chemical shifts had been sequentially assigned previously using principally 3D 1H-15N NOESY-HMQC and TOCSY-HMQC experiments [Fairbrother, W. J., Cavanagh, J., Dyson, H. J., Palmer, A. G., III, Sutrina, S. L., Reizer, J., Saier, M. H., Jr., & Wright, P. E. (1991) Biochemistry 30, 6896-6907]. The side-chain spin systems were identified using 3D HCCH-COSY and HCCH-TOCSY spectra and were assigned sequentially on the basis of their 1H alpha and 13C alpha chemical shifts. The 3D HCCH and HCA(CO)N experiments rely on large heteronuclear one-bond J couplings for coherence transfers and therefore offer a considerable advantage over conventional 1H-1H correlation experiments that rely on 1H-1H 3J couplings, which, for proteins the size of IIAglc (17.4 kDa), may be significantly smaller than the 1H line widths. The assignments reported herein are essential for the determination of the high-resolution solution structure of the IIAglc domain of B. subtilis using 3D and 4D heteronuclear edited NOESY experiments; these assignments have been used to analyze 3D 1H-15N NOESY-HMQC and 1H-13C NOESY-HSQC spectra and calculate a low-resolution structure [Fairbrother, W. J., Gippert, G. P., Reizer, J., Saier, M. H., Jr., & Wright, P. E. (1992) FEBS Lett. 296, 148-152].     

Stereochemistry of the concerted enolization catalyzed by delta 5-3-ketosteroid isomerase

The reaction catalyzed by delta 5-3-ketosteroid isomerase has been shown to occur via the concerted enolization of the delta 5-3-ketosteroid substrate to form a dienolic intermediate, brought about by Tyr-14, which hydrogen bonds to and protonates the 3-keto group, and Asp-38, which removes and axial (beta) proton from C-4 of the substrate, in the same rate-limiting step [Xue, L., Talalay, P., & Mildvan, A.S. (1990) Biochemistry 29, 7491-7500; Kuliopulos, A., Mildvan, A.S., Shortle, D., & Talalay, P. (1989) Biochemistry 26, 3927-3937]. Since the axial C-4 proton is removed by Asp-38 from above the substrate, a determination of the complete stereochemistry of this rapid, concerted enolization requires information on the direction of approach of Tyr-14 to the enzyme-bound steroid. The double mutant enzyme, Y55F + Y88F, which retains Tyr-14 as the sole Tyr residue, was prepared and showed only a 4.5-fold decrease in kcat (12,000 s-1) and a 3.6-fold decrease in KM (94 microM) for delta 5-androstene-3, 17,dione, in comparison with the wild-type enzyme. Deuteration of the aromatic rings of the 10 Phe residues further facilitated the assignment of the aromatic proton resonances of Tyr-14 in the 600-MHz TOCSY spectrum at 6.66 +/- 0.01 ppm (3,5H) and at 6.82 +/- 0.01 ppm (2,6H). Variation of the pH from 4.9 to 10.9 did not alter these shifts, indicating that the pKa of Tyr-14 exceeds 10.9. Resonances assigned to the three His residues titrated with pKa values very similar to those found with the wild-type enzyme. The binding of 19-nortestosterone, a product analogue and substrate of the reverse isomerase reaction, induced downfield shifts of -0.12 and -0.06 ppm of the 3,5-and 2,6-proton resonances of Tyr-14, respectively, possibly due to deshielding by the 3-keto group of the steroid, but also induced +0.29 to -0.41 ppm changes in the chemical shifts of 8 of the 10 Phe residues and smaller changes in 10 of the 12 ring-shifted methyl resonances, indicating a steroid-induced conformation change in the enzyme. NOESY spectra in H2O revealed strong negative Overhauser effects from the 3,5-proton resonance of Tyr-14 to the overlapping 2 alpha-, 2 beta-, or 6 beta-proton resonances of the bound steroid but no NOE's to the 4- or 6 alpha-protons of the steroid.(ABSTRACT TRUNCATED AT 400 WORDS)     

Multinuclear magnetic resonance studies of the 2Fe.2S* ferredoxin from Anabaena species strain PCC 7120. 2. Sequence-specific carbon-13 and nitrogen-15 resonance assignments of the oxidized form

Multinuclear two-dimensional NMR techniques were used to assign nearly all diamagnetic 13C and 15N resonances of the plant-type 2Fe.2S* ferredoxin from Anabaena sp. strain PCC 7120. Since a 13C spin system directed strategy had been used to identify the 1H spin systems [Oh, B.-H., Westler, W. M., & Markley, J. L. (1989) J. Am. Chem. Soc. 111, 3083-3085], the sequence-specific 1H assignments [Oh, B.-H., & Markley, J. L. (1990) Biochemistry (first paper of three in this issue)] also provided sequence-specific 13C assignments. Several resonances from 1H-13C groups were assigned independently of the 1H assignments by considering the distances between these nuclei and the paramagnetic 2Fe.2S* center. A 13C-15N correlation data set was used to assign additional carbonyl carbons and to analyze overlapping regions of the 13C-13C correlation spectrum. Sequence-specific assignments of backbone and side-chain nitrogens were based on 1H-15N and 13C-15N correlations obtained from various two-dimensional NMR experiments.     

Assignment of the side-chain 1H and 13C resonances of interleukin-1 beta using double- and triple-resonance heteronuclear three-dimensional NMR spectroscopy

The assignment of the aliphatic 1H and 13C resonances of IL-1 beta, a protein of 153 residues and molecular mass 17.4 kDa, is presented by use of a number of novel three-dimensional (3D) heteronuclear NMR experiments which rely on large heteronuclear one-bond J couplings to transfer magnetization and establish through-bond connectivities. These 3D NMR experiments circumvent problems traditionally associated with the application of conventional 2D 1H-1H correlation experiments to proteins of this size, in particular the extensive chemical shift overlap which precludes the interpretation of the spectra and the reduced sensitivity arising from 1H line widths that are often significantly larger than the 1H-1H J couplings. The assignment proceeds in two stages. In the first step the 13C alpha chemical shifts are correlated with the NH and 15N chemical shifts by a 3D triple-resonance NH-15N-13C alpha (HNCA) correlation experiment which reveals both intraresidue NH(i)-15N(i)-13C alpha (i) and some weaker interresidue NH(i)-15N(i)-C alpha (i-1) correlations, the former via intraresidue one-bond 1JNC alpha and the latter via interresidue two-bond 2JNC alpha couplings. As the NH, 15N, and C alpha H chemical shifts had previously been sequentially assigned by 3D 1H Hartmann-Hahn 15N-1H multiple quantum coherence (3D HOHAHA-HMQC) and 3D heteronuclear 1H nuclear Overhauser 15N-1H multiple quantum coherence (3D NOESY-HMQC) spectroscopy [Driscoll, P.C., Clore, G.M., Marion, D., Wingfield, P.T., & Gronenborn, A.M. (1990) Biochemistry 29, 3542-3556], the 3D triple-resonance HNCA correlation experiment permits the sequence-specific assignments of 13C alpha chemical shifts in a straightforward manner. The second step involves the identification of side-chain spin systems by 3D 1H-13C-13C-1H correlated (HCCH-COSY) and 3D 1H-13C-13C-1H total correlated (HCCH-TOCSY) spectroscopy, the latter making use of isotropic mixing of 13C magnetization to obtain relayed connectivities along the side chains. Extensive cross-checks are provided in the assignment procedure by examination of the connectivities between 1H resonances at all the corresponding 13C shifts of the directly bonded 13C nuclei. In this manner, we were able to obtain complete 1H and 13C side-chain assignments for all residues, with the exception of 4 (out of a total of 15) lysine residues for which partial assignments were obtained. The 3D heteronuclear correlation experiments described are highly sensitive, and the required set of three 3D spectra was recorded in only 1 week of measurement time on a single uniformly 15N/13C-labeled 1.7 mM sample of interleukin-1 beta.(ABSTRACT TRUNCATED AT 400 WORDS)     

31P NMR saturation-transfer and 13C NMR kinetic studies of glycolytic regulation during anaerobic and aerobic glycolysis

31P NMR saturation-transfer techniques have been employed in glucose-grown derepressed yeast to determine unidirectional fluxes in the upper part of the Embden-Meyerhof-Parnas pathway. The experiments were performed during anaerobic and aerobic glycolysis by saturating the ATP gamma resonances and monitoring changes in the phosphomonoester signals from glucose 6-phosphate and fructose 1,6-bis-phosphate. These experiments were supplemented with 13C NMR measurements of glucose utilization rates and 13C NMR label distribution studies. Combined with data obtained previously from radioisotope measurements, these 31P and 13C NMR kinetic studies allowed estimation of the net glycolytic flow in addition to relative flows through phosphofructokinase (PFK) and Fru-1,6-P2ase during anaerobic and aerobic glycolysis. The 31P NMR saturation-transfer results are consistent with previous results obtained from measurements of metabolite levels, radioisotope data, and 13C NMR studies [den Hollander, J.A., Ugurbil, K., Brown, T.R., Bednar, M., Redfield, C., & Shulman, R.G. (1986a) Biochemistry 25, 203-211], providing additional support for in vivo measurement of the flows during glycolysis.     

Direct observation of the enzyme-intermediate complex of 5-enolpyruvylshikimate-3-phosphate synthase by 13C NMR spectroscopy

The interaction of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, 4,5-dideoxyshikimate 3-phosphate (ddS3P), and [2-13C]-and [3-13C]phosphoenolpyruvate (PEP) has been examined by 13C NMR spectroscopy. Although no resonances due to a dead-end intermediate complex could be detected, an enzyme active site specific formation of pyruvate was observed. The interaction of EPSP synthase with shikimate 3-phosphate (S3P) and [2-13C]- or [3-13C]PEP has been examined by 13C NMR spectroscopy. With [2-13C]PEP, in addition to the resonances due to [2-13C]PEP and [8-13C]EPSP, new resonances appeared at 164.8, 110.9, and 107.2 ppm. The resonance at 164.8 ppm has been assigned to enzyme-bound EPSP. The resonance at 110.9 ppm has been assigned to C-8 of an enzyme-free tetrahedral intermediate of the sort originally proposed by Levin and Sprinson [Levin, J. G., & Sprinson, D. B. (1964) J. Biol. Chem. 239, 1142-1150] and recently independently observed by Anderson et al. [Anderson, K. S., Sikorski, J. A., Benesi, A. J., & Johnson, K. A. (1988) J. Am. Chem. Soc. 110, 6577-6579]. The resonance at 107.2 ppm has been assigned to an enzyme-bound intermediate whose structure is closely related to that of the tetrahedral intermediate. With [3-13C]PEP, new resonances appeared at 88.9, 26.2, 25.5, and 24.5 ppm. The resonance at 88.9 ppm has been assigned to enzyme-bound EPSP. The resonance at 26.2 ppm, which was found to correlate with 1.48 ppm by isotope-edited multiple quantum coherence 1H NMR spectroscopy, has been assigned to the methyl group 4-hydroxy-4-methylketoglutarate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Paramagnetic ion effects on the nuclear magnetic resonance spectrum of transfer ribonucleic acid: assignment of the 15--48 tertiary resonance.

We have studied the effects of Co2+ and Mn2+ ions on the low-field nuclear magnetic resonance (NMR) spectra of pure class 1 transfer ribonucleic acid (tRNA) species. With 1.2 mM tRNA in the presence of 15 mM MgCl2 discrete paramagnetic effects were observed for Co2+ at concentrations in the range 0.02--0.1 mM and for Mn2+ in the range 0.002--0.01 mM, indicating fast exchange of these cations with tRNA. Both of these cations paramagnetically relax the s4U8--A14 resonance as well as other resonances from proximal base pairs. The Co2+ site appears to be the same site on G15 which was observed crystallographically [Jack, A., Ladner, J. E., Rhodes, D., Brown, R. S., & Klug, A. (1977) J. Mol. Biol. 111, 315-328]; the initially occupied tight Mn2+ site is the cation site involving the phosphate of U8. There are three base pairs within 10 A of both sites, namely, G15--C48, A14--s4U8, and C13--G22; this has led to the assignment of the G15--C48 and C13--G22 resonances in the NMR spectrum [Jack, A., Ladner, J. E., Rhodes, D., Brown, R. S., & Klug, A. (1977) J. Mol. Biol. 111, 315--328; Holbrook, S. R., Sussman, J. L., Warrant, R. W., Church, G. M., & Kim, Sung-Hou (1977) Nucleic Acids Res. 4, 2811--2820; Quigley, G. J., Teeter, M. M., & Rich, A. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 64--68].     

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.     

Structural comparison of monomeric variants of the chemokine MIP-1beta having differing ability to bind the receptor CCR5

MIP-1beta, a member of the chemokine family of proteins, tightly binds the receptor CCR5 as part of its natural function in the immune response, and in doing so also blocks the ability of many strains of HIV to enter the cell. The single most important MIP-1beta residue known to contribute to its interaction with the receptor is Phe13, which when mutated reduces the ability of MIP-1beta to bind to CCR5 by more than 1000-fold. To obtain a structural understanding of the dramatic effect of the absence of Phe13 in MIP-1beta, we used multidimensional heteronuclear NMR to determine the three-dimensional structure of the MIP-1beta F13A variant. We had previously shown that, unlike the wild-type protein which has been shown to be a tight dimer, the F13A mutant is monomeric even at high concentrations [Laurence, J. S., Blanpain, C., Burgner, J. W., Parmentier, M., and LiWang, P. J. (2000) Biochemistry 39, 3401-3409], leading to significant changes in the NMR spectra of F13A and the wild-type protein. We have obtained a total of 940 structural restraints for MIP-1beta F13A, and have calculated a family of structures having a backbone rmsd from the average of 0.55 A (residues 12-67). A structural comparison of the F13A mutant with a fully active monomeric variant, P8A, shows that despite some differences in the (1)H-(15)N HSQC spectra the two are nearly identical in NOE distance restraints and in backbone conformation. A comparison of F13A with the wild-type protein shows largely the same fold, although differences exist in the N-terminal and loop regions for which the loss of the dimer in F13A can mainly account. A dynamics comparison confirms greater flexibility in F13A than in the wild-type protein in regions of dimer contact in the wild-type protein. In an analysis to determine if the large functional effect resulting from the loss of Phe13 is due to the local side chain change or due to more global structural changes, we conclude that local effects predominate. This suggests that a strategy for designing tight binding anti-CCR5 therapeutics should include a Phe-like component.     

NMR studies of the association of cytochrome b5 with cytochrome c

In an effort to gain greater insight into the molecular mechanism of the electron-transfer reactions of cytochrome b(5), the bovine cytochrome b(5)-horse cytochrome c complex has been investigated by high-resolution multidimensional NMR spectroscopy using (13)C, (15)N-labeled cytochrome b(5) expressed from a synthetic gene. Chemical shifts of the backbone (15)N, (1)H, and (13)C resonances for 81 of the 82 residues of [U-90% (13)C,U-90% (15)N]-ferrous cytochrome b(5) in a 1:1 complex with ferrous cytochrome c were compared with those of ferrous cytochrome b(5) in the absence of cytochrome c. A total of 51% of these residues showed small, but significant, changes in chemical shifts (the largest shifts were 0.1 ppm for the amide (1)H, 1.15 for (13)C(alpha), 1.03 ppm for the amide (15)N, and 0.15 ppm for the (1)H(alpha) resonances). Some of the residues exhibiting chemical shift changes are located in a region that has been implicated as the binding surface to cyt c [Salemme, F. R. (1976) J. Mol. Biol. 10, 563-568]. Surprisingly, many of the residues with changes are not located on this surface. Instead, they are located within and around a cleft observed to form in a molecular dynamics study of cytochrome b(5) [Storch, E. M., and Daggett, V. (1995) Biochemistry 34, 9682-9693](.) The rim of this cleft can readily accommodate cytochrome c. Molecular dynamics simulations of the Salemme and cleft complexes were performed for 2 ns and both complexes were stable.     

H-1, C-13, and F-19 nuclear magnetic resonance assignments of 3,3-difluoro-5 alpha-androstane-17 beta-ol acetate.

The molecular structure of 3,3-difluoro-5 alpha-androstane-17 beta-ol acetate was analyzed by 1H, 13C, and 19F nuclear magnetic resonance (NMR) techniques; two-dimensional NMR was used to assigned 1H and 13C resonances. The 1H NMR spectrum in deuterated chloroform shows three sharp singlets (delta = 0.74, 0.79, and 2.00 ppm) integrating for three protons each, an isolated triplet at 4.55 ppm integrating for one proton, and overlapping multiplets between 0.72 and 2.12 ppm integrating for 31 protons. The 13C spectrum shows 18 resonances between 10 and 55 ppm, and three additional resonances at 82.9, 124.0, and 171.5 ppm. The 19F[1H] spectrum shows two sets of doublets (observed 2J = 150 Hz) at 5.00 and -4.80 ppm. Multiplets arising from 19F-13C J-coupling provide the starting assignment for all resonances by means of 1H homonuclear correlation (COSY) and 1H-13C heteronuclear correlation spectroscopy.