Characterization of hydride transfer to flavin adenine dinucleotide in neuronal nitric oxide synthase reductase domain: geometric relationship between the nicotinamide and isoalloxazine rings.

Based on the similarity in both structure and function of the reductase domain of neuronal nitric oxide synthase (nNOSred) to that of NADPH-cytochrome P450 reductase (CPR), we determined whether the characteristics of hydride transfer from NADPH to flavin adenine dinucleotide (FAD) were similar for both proteins. Secondly, we questioned whether hydride transfer from NADPH to either nNOSred or holo-nNOS was rate limiting for reactions catalyzed by these two proteins. Utilizing 500 MHz proton NMR and deuterated substrate, we determined that the stereospecificity of hydride transfer from NADPH and the conformation of the nicotinamide ring around the glycosidic bond were similar between CPR and nNOSred. Specifically, nNOSred abstracts the A-side hydrogen from NADPH, and the nicotinamide ring is in the anti conformation. We determined that the rate of hydride transfer to FAD appears to become partially rate limiting only for exceptionally good electron acceptors such as cytochrome c. Hydride transfer is not rate limiting for NO. production under any conditions used in this study. Interestingly, the deuterium isotope effect was decreased in the cytochrome c reductase assay with both nNOS and nNOSred when the assays were conducted in high ionic strength buffer, suggesting an increase in the rate of hydride transfer to FAD. These results are in stark contrast to results obtained with CPR (D. S. Sem and C. B. Kasper, 1995, Biochemistry 34, 3391-3398) whereby hydride transfer is partially rate limiting at high, but not at low, ionic strength. The seemingly opposite results in deuterium isotope effect observed with CPR and nNOSred, under conditions of high and low ionic strength, suggest differences in structure and/or regulation of these important flavoproteins.

Complexes with truncated RNAs from the large domain of Archaeoglobus fulgidus signal recognition particle.

Protein SRP19 is an important component of the signal recognition particle (SRP) as it promotes assembly of protein SRP54 with SRP RNA and recognizes a tetranucleotide loop. Structural features and RNA binding activities of SRP19 of the hyperthermophilic archaeon Archaeoglobus fulgidus were investigated. An updated alignment of SRP19 sequences predicted three conserved regions and two alpha-helices. With Af-SRP RNA the Af-SRP54 protein assembled into an A. fulgidus SRP which remained intact for many hours. Stable complexes were formed between Af-SRP19 and truncated SRP RNAs, including a 36-residue fragment representing helix 6 of A. fulgidus SRP RNA.

High resolution solution structure of ribosomal protein L11-C76, a helical protein with a flexible loop that becomes structured upon binding to RNA.

The structure of the C-terminal RNA recognition domain of ribosomal protein L11 has been solved by heteronuclear three-dimensional nuclear magnetic resonance spectroscopy. Although the structure can be considered high resolution in the core, 15 residues between helix alpha 1 and strand beta 1 form an extended, unstructured loop. 15N transverse relaxation measurements suggest that the loop is moving on a picosecond-to-nanosecond time scale in the free protein but not in the protein bound to RNA. Chemical shifts differences between the free protein and the bound protein suggest that the loop as well as the C-terminal end of helix alpha 3 are involved in RNA binding.

The RNA binding domain of ribosomal protein L11: three-dimensional structure of the RNA-bound form of the protein and its interaction with 23 S rRNA.

The three-dimensional solution structure has been determined by NMR spectroscopy of the 75 residue C-terminal domain of ribosomal protein L11 (L11-C76) in its RNA-bound state. L11-C76 recognizes and binds tightly to a highly conserved 58 nucleotide domain of 23 S ribosomal RNA, whose secondary structure consists of three helical stems and a central junction loop. The NMR data reveal that the conserved structural core of the protein, which consists of a bundle of three alpha-helices and a two-stranded parallel beta-sheet four residues in length, is nearly the same as the solution structure determined for the non-liganded form of the protein. There are however, substantial chemical shift perturbations which accompany RNA binding, the largest of which map onto an extended loop which bridges the C-terminal end of alpha-helix 1 and the first strand of parallel beta-sheet. Substantial shift perturbations are also observed in the N-terminal end of alpha-helix 1, the intervening loop that bridges helices 2 and 3, and alpha-helix 3. The four contact regions identified by the shift perturbation data also displayed protein-RNA NOEs, as identified by isotope-filtered three-dimensional NOE spectroscopy. The shift perturbation and NOE data not only implicate helix 3 as playing an important role in RNA binding, but also indicate that regions flanking helix 3 are involved as well. Loop 1 is of particular interest as it was found to be flexible and disordered for L11-C76 free in solution, but not in the RNA-bound form of the protein, where it appears rigid and adopts a specific conformation as a result of its direct contact to RNA.

Binding affinity of transforming growth factor-beta for its type II receptor is determined by the C-terminal region of the molecule.

Transforming growth factor-beta (TGF-beta) isoforms have differential binding affinities for the TGF-beta type II receptor (TbetaRII). In most cells, TGF-beta1 and TGF-beta3 bind to TbetaRII with much higher affinity than TGF-beta2. Here, we report an analysis of the effect of TGF-beta structure on its binding to TbetaRII by using TGF-beta mutants with domain deletions, amino acid replacements, and isoform chimeras. Examination of the binding of TGF-beta mutants to the recombinant extracellular domain of TbetaRII by a solid-phase TGF-beta/TbetaRII assay demonstrated that only those TGF-beta mutants containing the C terminus of TGF-beta1 (TGF-beta1-(Delta69-73), TGF-beta1-(Trp71), and TGF-beta2/beta1-(83-112)) bind with high affinity to TbetaRII, similar to native TGF-beta1. Moreover, replacement of only 6 amino acids in the C terminus of TGF-beta1 with the corresponding sequence of TGF-beta2 (TGF-beta1/beta2-(91-96)) completely eliminated the high affinity binding of TGF-beta1. Proliferation of fetal bovine heart endothelial (FBHE) cells was inhibited to a similar degree by all of the TGF-beta mutants. However, recombinant soluble TbetaRII blocked the inhibition of FBHE cell proliferation induced by TGF-beta mutants retaining the C terminus of TGF-beta1, consistent with the high binding affinity between these TGF-beta molecules and TbetaRII. It was further confirmed that the TGF-beta2 mutant with its C terminus replaced by that of TGF-beta1 (TGF-beta2/beta1-(83-112)) competed as effectively as TGF-beta1 with 125I-TGF-beta1 for binding to membrane TbetaRI and TbetaRII on FBHE cells. These observations clearly indicate that the domain in TGF-beta1 responsible for its high affinity binding to TbetaRII, both the soluble and membrane-bound forms, is located at C terminus of the molecule.

Engineered disulfide bonds in staphylococcal nuclease: effects on the stability and conformation of the folded protein.

Efforts to enhance the stability of proteins by introducing engineered disulfide bonds have resulted in mixed success. Most approaches to the prediction of the energetic consequences of disulfide bond formation in proteins have considered only the destabilizing effects of cross-links on the unfolded state (chain entropy model) [Pace, C. N., Grimsley, G. R., Thomson, J. A., & Barnett, B. J. (1988) J. Biol. Chem. 263, 11820-11825: Doig, A. J., & Williams, D. H. (1991) J. Mol. Biol. 217, 389-398]. It seems clear, however, that disulfide bridges also can influence the stability of the native state. In order to assess the importance of the latter effect, we have studied four variants of staphylococcal nuclease (V8 strain) each containing one potential disulfide bridge created by changing two wild-type residues to cysteines by site-directed mutagenesis. In each case, one of the introduced cysteines was within the type VIa beta turn containing cis Pro117, and the other was located in the adjacent extended loop containing Gly79. In all four cases, the overall loop size was kept nearly constant (the number of residues in the loop between the two cysteines varied from 37 to 42) so as to minimize differences from chain entropy effects. The objective was to create variants in which a change in the reduction state of the disulfide would be coupled to a change in the position of the equilibrium between the cis and trans forms of the Xxx116-Pro117 peptide bond in the folded state of the protein. The position of this equilibrium, which can be detected by NMR spectroscopy, has been shown previously to correlate with the stability of the native protein. Its determination provides a measure of strain in the folded state. The thermal stabilities and free energies for unfolding by elevated temperature and guanidinium chloride were measured for each of the four mutants under conditions in which the introduced cysteines were cross-linked (oxidized) and unlinked (reduced). In addition, reduction potentials were determined for each mutant. Formation of the different disulfide bridges was found to induce varying levels of folded state strain. The stabilization energy of a given disulfide bridge could be predicted from the measured perturbation energy for the peptide bond isomerization, provided that energetic effects on the unfolded state were calculated according to the chain entropy model. Undiagnosed strain in native states of proteins may explain the variability observed in the stabilization provided by engineered disulfide bridges.

Transforming growth factor beta 1: three-dimensional structure in solution and comparison with the X-ray structure of transforming growth factor beta 2.

The three-dimensional solution structure of human transforming growth factor beta 1 (TGF-beta 1) has been determined using multinuclear magnetic resonance spectroscopy and a hybrid distance geometry/ simulated annealing algorithm. It represents one of the first examples of a mammalian protein structure that has been solved by isotopic labeling of the protein in a eukaryotic cell line and multinuclear NMR spectroscopy. The solution structure of the 25 kDa disulfide-linked TGF-beta 1 homodimer was calculated from over 3200 distance and dihedral angle restraints. The final ensemble of 33 accepted structures had no NOE or dihedral angle violations greater than 0.30 A and 5.0 degrees, respectively. The RMSD of backbone atoms for the ensemble of 33 structures relative to their mean structure was 1.1 A when all residues were used in the alignment and 0.7 A when loop regions were omitted. The solution structure of TGF-beta 1 follows two independently determined crystal structures of TGF-beta 2 (Daopin et al., 1992, 1993; Schlunegger & Grütter, 1992, 1993), providing the first opportunity to examine structural differences between the two isoforms at the molecular level. Although the structures are very similar, with an RMSD in backbone atom positions of 1.4 A when loop regions are omitted in the alignment and 1.9 A when all residues are considered, there are several notable differences in structure and flexibility which may be related to function. The clearest example of these is in the beta-turn from residues 69-72: the turn type found in the solution structure of TGF-beta 1 falls into the category of type II, whereas that present in the X-ray crystal structure of TGF-beta 2 is more consistent with a type I turn conformation. This may be of functional significance as studies using TGF-beta chimeras and deletion mutants indicate that this portion of the molecule may be important in receptor binding.

Three-dimensional solution structure of the HIV-1 protease complexed with DMP323, a novel cyclic urea-type inhibitor, determined by nuclear magnetic resonance spectroscopy.

The three-dimensional solution structure of the HIV-1 protease homodimer, MW 22.2 kDa, complexed to a potent, cyclic urea-based inhibitor, DMP323, is reported. This is the first solution structure of an HIV protease/inhibitor complex that has been elucidated. Multidimensional heteronuclear NMR spectra were used to assemble more than 4,200 distance and angle constraints. Using the constraints, together with a hybrid distance geometry/simulated annealing protocol, an ensemble of 28 NMR structures was calculated having no distance or angle violations greater than 0.3 A or 5 degrees, respectively. Neglecting residues in disordered loops, the RMS deviation (RMSD) for backbone atoms in the family of structures was 0.60 A relative to the average structure. The individual NMR structures had excellent covalent geometry and stereochemistry, as did the restrained minimized average structure. The latter structure is similar to the 1.8-A X-ray structure of the protease/DMP323 complex (Chang CH et al., 1995, Protein Science, submitted); the pairwise backbone RMSD calculated for the two structures is 1.22 A. As expected, the mismatch between the structures is greatest in the loops that are disordered and/or flexible. The flexibility of residues 37-42 and 50-51 may be important in facilitating substrate binding and product release, because these residues make up the respective hinges and tips of the protease flaps. Flexibility of residues 4-8 may play a role in protease regulation by facilitating autolysis.

NMR strategy for determining Xaa-Pro peptide bond configurations in proteins: mutants of staphylococcal nuclease with altered configuration at proline-117.

A general approach has been developed for configurational analysis (cis or trans) of Xaa-Pro peptide bonds in proteins. This approach, which entails selective 13C labeling of Xaa and Pro residues in the protein and isotope-edited NMR, has been applied to mutants of staphylococcal nuclease with suspected altered configurations of the Lys116-Pro117 peptide bond. The technique for monitoring proline configurations is based on differences in interproton distances between the H alpha of residue Xaa and the proline H delta or H alpha protons. Short (< 2.5 A) Xaa H alpha-Pro H delta interproton distances are diagnostic for the trans configuration, whereas short (< 2.5 A) Xaa H alpha-Pro H alpha interproton distances are diagnostic for the cis configuration. Biosynthetic incorporation of [alpha-13C]Xaa and [delta-13C]proline facilitates detection of trans Xaa-Pro peptide bonds, whereas incorporation of [alpha-13C]Xaa and [alpha-13C]proline facilitates detection of cis Xaa-Pro peptide bonds. Provided that the Xaa-Pro peptide bond is unique within the protein sequence, symmetric off-diagonal NOE cross peaks in the isotope-edited NOE spectrum allow for simultaneous chemical shift assignment and determination of the prolyl peptide bond geometry. We have used this technique to determine the predominant configuration of the Lys116-Pro117 peptide bond in recombinant V8 staphylococcal nuclease A (H124L) and two of its single amino acid mutants (D77A+H124L and G79S+H124L). The results are consistent with conclusions reached on the basis of indirect arguments concerning changes in the chemical shifts of histidine 1H epsilon 1 NMR signals.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of amino acid substitutions on the pressure denaturation of staphylococcal nuclease as monitored by fluorescence and nuclear magnetic resonance spectroscopy.

In the present study we have used high hydrostatic pressure coupled with either time-resolved and steady-state fluorescence or NMR spectroscopy in order to investigate the effects of amino acid substitutions on the high-pressure denaturation properties of staphylococcal nuclease. This protein has been shown previously to be structurally heterogeneous in its native state. On the NMR time scale, four distinct interconverting conformational forms arise from the population of both cis and trans Xaa-Pro peptide bonds (His46-Pro47 and Lys116-Pro117) [Evans et al. (1989) Biochemistry 28, 362; Loh et al. (1991) in Techniques in Protein Chemistry II, pp 275-282, Academic Press, New York]. Mutations in the protein sequence have been shown to change the distribution among the various forms [Alexandrescu et al. (1989) Biochemistry 28, 204; Alexandrescu et al. (1990) Biochemistry 29, 4516]. Time-resolved fluorescence on a series of mutants with altered equilibria for cis/trans isomerism about the 116-117 peptide bond did not reveal any simple relationship between the position of the cis/trans equilibrium in the folded state and the heterogeneity of the fluorescence decay. However, the specific dynamic properties of each mutant, as revealed by time-resolved fluorescence, do appear to be correlated with their partial molar volume changes of denaturation. A striking finding is that mutation of either (or both) of the prolines that exhibits structural heterogeneity to glycine greatly alters the stability of the protein to pressure. These mutations also result in decreased chain mobility as assessed by time-resolved fluorescence. It appears that packing defects, which allow for peptide bond cis/trans heterogeneity in the wild-type protein, are removed by the Pro-->Gly substitutions.

Overexpression and purification of avian ovomucoid third domains in Escherichia coli.

Genetic engineering studies of ovomucoid domains have been hindered by the lack of an efficient procedure for overproducing this protein. The novel scheme presented here has led to the isolation of chicken ovomucoid third domain (OMCHI3) at a level of 22 mg pure protein/l Escherichia coli culture medium. The gene coding for OMCHI3 was fused to the 3' end of the gene encoding staphylococcal nuclease (SNase). Expression of the chimeric gene was placed under control of the strong transcription and translation signals of the phage T7 promoter. Upon isopropyl-beta-D-galactopyranoside induction, the cells harboring the target plasmid efficiently overproduced the protein (30% of the total soluble protein). The 56-residue fragment corresponding to OMCHI3 was then liberated by cyanogen bromide (CNBr) cleavage at a genetically engineered methionine residue located at the nuclease--OMCHI3 junction (OMCHI3 lacks an internal methionine). SDS--PAGE, enzyme inhibition studies and NMR spectroscopy all indicated that the recombinant OMCHI3 has properties identical to those of OMCHI3 isolated from its natural source. The expression system was easily adapted for the production of [98% U 15N] OMCHI3. The expression vector was mutated for overexpression of turkey ovomucoid third domain (OMTKY3), which differs from OMCHI3 by three amino acid substitutions. Since many other avian ovomucoid domains also lack methionine residues, this approach should be suitable for large-scale production and isotope labeling of homologous proteinase inhibitors with a variety of inhibitory specificities.

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.

Coupling between local structure and global stability of a protein: mutants of staphylococcal nuclease.

Staphylococcal nuclease exists in solution as a mixture of two folded (N and N') and two unfolded (U and U*) forms. Earlier workers [Evans et al. (1989) Biochemistry 28, 362] have proposed that the N'/N and U/U* structural differences involve cis/trans isomerization about the Lys116-Pro117 peptide bond with N and U cis and N' and U* trans. The present results show that residue changes throughout the nuclease structure have large effects on the distribution of the N and N'forms. The N'/N ratios at 313 K for nuclease H124L (N'/N = 0.07) and nuclease G79S (N'/N = 12) differ by 2 orders of magnitude. Thermodynamic parameters for equilibria linking the two folded and two unfolded substates were evaluated for seven mutants of nuclease which were found by kinetic assays to have similar enzymatic activities but by NMR spectroscopy to have a wide dispersion of thermal stabilities. Our results indicate that mutational perturbations of the N'/N equilibrium in folded nuclease (delta G for the N in equilibrium N' reaction) are strongly coupled to changes in the stability of the N form (delta G for the N in equilibrium U reaction), but much less so to the stability of the N' form (delta G for the N' in equilibrium U* reaction).

Two-dimensional NMR studies of staphylococcal nuclease: evidence for conformational heterogeneity from hydrogen-1, carbon-13, and nitrogen-15 spin system assignments of the aromatic amino acids in the nuclease H124L-thymidine 3',5'-bisphosphate-Ca2+ ternary complex.

A combination of multinuclear two-dimensional NMR experiments served to identify and assign the combined 1H, 13C, and 15N spin systems of the single tryptophan, three phenylalanines, three histidines, and seven tyrosines of staphylococcal nuclease H124L in its ternary complex with calcium and thymidine 3',5'-bisphosphate at pH 5.1 (H2O) or pH 5.5 (2H2O). Samples of recombinant nuclease were labeled with 13C or 15N as appropriate to individual NMR experiments: uniformly with 15N (all sites to greater than 95%), uniformly with 13C (all sites to 26%), selectively with 13C (single amino acids uniformly labeled to 26%), or selectively with 15N (single amino acids uniformly labeled to greater than 95%). NMR data used in the analysis included single-bond and multiple-bond 1H-13C and multiple-bond 1H-15N correlations, 1H-13C single-bond correlation with Hartmann-Hahn relay (1H[13C]SBC-HH), and 1H-13C single-bond correlation with NOE relay (1H[13C]SBC-NOE). The aromatic protons of the spin systems were identified from 1H[13C]SBC-HH data, and the nonprotonated aromatic ring carbons were identified from 1H-13C multiple-bond correlations. Sequence-specific assignments were made on the basis of observed NOE relay connectivities between assigned 1H alpha-13C alpha or 1H beta-13C beta direct cross peaks in the aliphatic region [Wang, J., LeMaster, D. M., & Markley, J. L. (1990) Biochemistry 29, 88-101] and 1H delta-13C delta direct cross peaks in the aromatic region of the 1H[13C]SBC-NOE spectrum. The His121 1H delta 2 resonance, which has an unusual upfield shift (at 4.3 ppm in the aliphatic region), was assigned from 1H[13C]SBC, 1H[13C]MBC, and 1H[15N]MBC data. Evidence for local structural heterogeneity in the ternary complex was provided by doubled peaks assigned to His46, one tyrosine, and one phenylalanine. Measurement of NOE buildup rates between protons on different aromatic residues of the major ternary complex species yielded a number of interproton distances that could be compared with those from X-ray structures of the wild-type nuclease ternary complex with calcium and thymidine 3',5'-bisphosphate [Cotton, F. A., Hazen, E. E., Jr., & Legg, M. J. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 2551-2555; Loll, P. J., & Lattman, E. E. (1989) Proteins: Struct., Funct., Genet. 5, 183-201]. The unusual chemical shift of His121 1H delta 2 is consistent with ring current calculations from either X-ray structure.

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)].