Two-dimensional NMR studies of staphylococcal nuclease. 2. Sequence-specific assignments of carbon-13 and nitrogen-15 signals from the nuclease H124L-thymidine 3',5'-bisphosphate-Ca2+ ternary complex

Samples of staphylococcal nuclease H124L (cloned protein overproduced in Escherichia coli whose sequence is identical with that of the nuclease isolated from the V8 strain of Staphylococcus aureus) were labeled uniformly with carbon-13 (26% ul 13C), uniformly with nitrogen-15 (95% ul 15N), and specifically by incorporating nitrogen-15-labeled leucine ([98% 15N]Leu) or carbon-13-labeled lysine ([26% ul 13C]Lys), arginine ([26% ul 13C]Arg), or methionine ([26% ul 13C]Met). Solutions of the ternary complexes of these analogues (nuclease H124L-pdTp-Ca2+) at pH 5.1 (H2O) or pH* 5.5 (2H2O) at 45 degrees C were analyzed as appropriate to the labeling pattern by multinuclear two-dimensional (2D) NMR experiments at spectrometer fields of 14.09 and 11.74 T: 1H-13C single-bond correlation (1H[13C]SBC); 1H-13C single-bond correlation with NOE relay (1H[13C]SBC-NOE); 1H-13C single-bond correlation with Hartmann-Hahn relay (1H-[13C]SBC-HH); 1H-13C multiple-bond correlation (1H[13C]MBC); 1H-15N single-bond correlation (1H-[15N]SBC); 1H-15N single-bond correlation with NOE relay (1H[15N]SBC-NOE). The results have assisted in spin system assignments and in identification of secondary structural elements. Nuclear Overhauser enhancements (NOE's) characteristic of antiparallel beta-sheet (d alpha alpha NOE's) were observed in the 1H [13C]-SBC-NOE spectrum of the nuclease ternary complex labeled uniformly with 13C. NOE's characteristic of alpha-helix (dNN NOE's) were observed in the 1H[15N]SBC-NOE spectrum of the complex prepared from protein labeled uniformly with 15N. The assignments obtained from these multinuclear NMR studies have confirmed and extended assignments based on 1H[1H] 2D NMR experiments [Wang, J., LeMaster, D. M., & Markley, J. L. (1990) Biochemistry (preceding paper in this issue)].     

Two-dimensional NMR studies of staphylococcal nuclease. 1. Sequence-specific assignments of hydrogen-1 signals and solution structure of the nuclease H124L-thymidine 3',5'-bisphosphate-Ca2+ ternary complex

Staphylococcal nuclease H124L is a recombinant protein produced in Escherichia coli whose sequence is identical with that of the nuclease produced by the V8 variant of Staphylococcus aureus. The enzyme-metal ion activator-nucleotide inhibitor ternary complex, nuclease H124L-thymidine 3',5'-bisphosphate-Ca2+, was investigated by two-dimensional (2D) NMR techniques. Efficient overproduction of the enzyme facilitated the production of random fractionally deuterated protein, which proved essential for detailed NMR analysis. 1H NMR spin systems were analyzed by conventional 2D 1H[1H] methods: COSY, relayed COSY, HOHAHA, and NOESY. Assignments obtained by 1H NMR experiments were confirmed and extended by 1H-13C and 1H-15N heteronuclear NMR experiments [Wang, J., Hinck, A. P., Loh, S. N., & Markley, J. L. (1990) Biochemistry (following paper in this issue)]. Spectra of the ternary complexes prepared with protein at natural abundance and at 50% random fractional deuteration provided the information needed for sequence-specific assignments of 121 of the 149 amino acid residues. Short- and intermediate-range NOE connectivities allowed the determination of secondary structural features of the ternary complex: three alpha-helical domains and three antiparallel beta-pleated sheets with several reverse turns. A number of nonsequential long-range HN-HN and H alpha-HN connectivities revealed additional information about the spatial arrangement of these secondary structural elements. The solution structure of this ternary complex shows a close correspondence to the crystal structure of the nuclease wt-thymidine 3',5'-bisphosphate-Ca2+ ternary complex [Cotton, F. A., Hazen, E. E., & Legg, M. J. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 2551-2555].     

Concerted two-dimensional NMR approaches to hydrogen-1, carbon-13, and nitrogen-15 resonance assignments in proteins

When used in concert, one-bond carbon-carbon correlations, one-bond and multiple-bond proton-carbon correlations, and multiple-bond proton-nitrogen correlations, derived from two-dimensional (2D) NMR spectra of isotopically enriched proteins, provide a reliable method of assigning proton, carbon, and nitrogen resonances. In contrast to procedures that simply extend proton assignments to carbon or nitrogen resonances, this technique assigns proton, carbon, and nitrogen resonances coordinately on the basis of their integrated coupling networks. Redundant spin coupling pathways provide ways of resolving overlaps frequently encountered in homonuclear 1H 2D NMR spectra and facilitate the elucidation of complex proton spin systems. Carbon-carbon and proton-carbon couplings can be used to bridge the aromatic and aliphatic parts of proton spin systems; this avoids possible ambiguities that may result from the use of nuclear Overhauser effects to assign aromatic amino acid signals. The technique is illustrated for Anabaena 7120 flavodoxin and cytochrome c-553, both uniformly enriched with carbon-13 (26%) or nitrogen-15 (98%).     

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.     

Backbone 1H, 13C, 15N NMR assignments of the unliganded and substrate ternary complex forms of mevalonate diphosphate decarboxylase from Streptococcus pneumoniae

Mevalonate diphosphate decarboxylase (MDD) catalyzes the ATP-dependent decarboxylation of diphosphomevalonate (DPM) to produce isopentenyl diphosphate (IPP), the molecular “building block” for more than 25,000 distinct isoprenoids, including cholesterol, steroid hormones and terpenoids. Here, we present the first backbone assignment of Streptococcus pneumoniae MDD in the unliganded state and in a ternary complex with DPM and AMPPCP––a nucleotide analogue unable to transfer the γ-phosphoryl group. The secondary chemical shifts for the unliganded form are in good agreement with the crystal structure of Streptococcus pyogenes (~70% sequence identity). The addition of substrate and nucleotide to the enzyme results in chemical shift changes of cross peaks that correspond to residues in the binding pocket.     

1H, 13C and 15N NMR assignments of the E. coli peptide deformylase in complex with a natural inhibitor called actinonin

In eubacteria, the formyl group of nascent polypeptides is removed by peptide deformylase protein (PDF). This is the reason why PDF has received special attention in the course of the search for new antibacterial agents. We observed by NMR that actinonin, a natural inhibitor, induced drastic changes in the HSQC spectrum of E. coli PDF. We report here the complete NMR chemical shift assignments of PDF resonances bound to actinonin.     

1H, 13C and 15N random coil NMR chemical shifts of the common amino acids. I. Investigations of nearest-neighbor effects

Summary In this study we report on the ¹H, ¹³C and ¹⁵N NMR chemical shifts for the random coil state and nearest-neighbor sequence effects measured from the protected linear hexapeptide Gly-Gly-X-Y-Gly-Gly (where X and Y are any of the 20 common amino acids). We present data for a set of 40 peptides (of the possible 400) including Gly-Gly-X-Ala-Gly-Gly and Gly-Gly-X-Pro-Gly-Gly, measured under identical aqueous conditions. Because all spectra were collected under identical experimental conditions, the data from the Gly-Gly-X-Ala-Gly-Gly series provide a complete and internally consistent set of ¹H, ¹³C and ¹⁵N random coil chemical shifts for all 20 common amino acids. In addition, studies were also conducted into nearest-neighbor effects on the random coil shift arising from a variety of X and Y positional substitutions. Comparisons between the chemical shift measurements obtained from Gly-Gly-X-Ala-Gly-Gly and Gly-Gly-X-Pro-Gly-Gly reveal significant systematic shift differences arising from the presence of proline in the peptide sequence. Similarly, measurements of the chemical shift changes occurring for both alanine and proline (i.e., the residues in the Y position) are found to depend strougly on the type of amino acid substituted into the X position. These data lend support to the hypothesis that sequence effects play a significant role in determining peptide and protein chemical shifts.     

Aliphatic 1H, 13C and 15N chemical shift assignments of dihydrofolate reductase from the psychropiezophile Moritella profunda in complex with NADP+ and folate

Dihydrofolate reductase from the deep-sea bacterium Moritella profunda (MpDHFR) has been ¹³C/¹⁵N isotopically labelled and purified. Here, we report the aliphatic ¹H, ¹³C and ¹⁵N resonance assignments of MpDHFR in complex with NADP⁺ and folate. The spectra of MpDHFR suggest considerably greater conformational heterogeneity than is seen in the closely related DHFR from Escherichia coli.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N NMR assignments of an unusual Ca<Superscript>2+</Superscript>-binding protein from <Emphasis Type="Italic">Entamoeba histolytica</Emphasis> in its apo form

We report almost complete sequence specific ¹H, ¹³C and ¹⁵N NMR assignments of an unusual Ca²⁺-binding protein from Entamoeba histolytica (EhCaBP6) in its apo form as a prelude to its structural and functional characterization.