DNA duplex dynamics: NMR relaxation studies of a decamer with uniformly 13C-labeled purine nucleotides

Dynamics in a DNA decamer duplex, d(CATTTGCATC). d(GATGCAAATG), were investigated via a detailed 13C NMR relaxation study. Every 2'-deoxyadenosine and 2'-deoxyguanidine was chemically enriched with 15% 13C and 98% 15N isotopes. Six nuclear relaxation parameters [R(13Cz), R(1Hz), R(2(1)Hz13Cz), R(13Cx), R(2(1)Hz13Cx) and steady-state 13C?1H? NOE] were measured at 600 MHz and three were measured at 500 MHz (1H frequency) for the CH spin systems of sugar 1', 3', and 4' as well as base 8 and 2 positions. A dependence of relaxation parameter values on chemical position was clearly observed; however, no sequence-specific variation was readily evident within our experimental error of approximately 5-10%, except for 3' and 5' termini. It was demonstrated that the random 15% 13C enrichment effectively suppressed both scalar and dipolar contributions of the neighboring carbons and protons on the relaxation parameters. To analyze dynamics via all observed relaxation parameters, full spectral density mapping (1992, J. W. Peng and G. Wagner, J. Magn. Reson. 98, 308) and the "model-free" approach (1982, Lipari and Szabo, J. Am. Chem. Soc. 104, 4546) were applied complementarily. A linear correlation between three spectral density values, J(omegaC), J(omegaH - omegaC), and J(omegaH + omegaC) was observed in plots containing all measured values, but not for the other spectral density terms including J(0). These linear correlations reflect the effect of overall motion and similar internal motions for each CH vector in the decamer. The correlations yielded two correlation times, 3-4 ns and 10-200 ps. One value, 3-4 ns, corresponds to the value of 3.3 ns obtained for the overall isotropic tumbling correlation time determined from analysis of 13C T1/T2 ratios. The possibility of overall anisotropic tumbling was examined, but statistical analysis showed no advantage over the assumption of simple isotropic tumbling. Lack of correlations entailing J(0) implies that a relatively slow chemical exchange contributes to yielding of effective Jeff(0) values. Based on spectral density mapping and the T1/T2 ratio analysis, three basic assumptions were initially employed (and subsequently justified) for the model-free calculation: isotropic overall tumbling, one internal motion, and the presence of chemical exchange terms. Except for terminal residues, the order parameter S2 and the corresponding fast internal motion correlation time were determined to be about 0.8 +/- 0.1 and 20 +/- 20 ps, respectively, for the various CH vectors. Only a few differences were observed between or within sugars and bases. The internal motion is very fast (ps-ns time scale) and its amplitude restricted; e.g., assuming a simple wobble-in-a-cone model, the internal motion is restricted to an angular amplitude of +/-22. 5 degrees for each of the 1', 3', 4', 2, and 8 positions in the purine nucleotides in the entire duplex.     

Resonance assignment of methionine methyl groups and chi 3 angular information from long-range proton-carbon and carbon-carbon J correlation in a calmodulin-peptide complex

Several simple 3D experiments are used to provide J correlations between methionine C epsilon methyl carbons and either the C gamma H2 protons or C beta and C gamma. The intensity of the J correlations provides information on the size of the three-bond J couplings and thereby on the chi 3 torsion angle. In addition, a simple 3D version of the HMBC experiment provides a sensitive link between the C epsilon H3 methyl protons and C gamma. The methods are demonstrated for a 20 kDa complex between calmodulin and a 26-residue peptide fragment of skeletal muscle myosin light chain kinase.     

Structural studies by high-field NMR spectroscopy of a binary-addressed complementary oligonucleotide system juxtaposing pyrene and perfluoro-azide units

Recently, a new approach has been proposed to improve the site-specificity and efficiency of the modification of nucleic acid target sequences, the binary system of complementary-addressing nucleic acid sequences. The binary system comprises two oligonucleotides, one modified with a photosensitizing group and the other with a photoreactive group. The sites of chemical modification are arranged to bring the two chemical functions close enough together in space to allow efficient energy transfer from the photo-excited photosensitizer to an arylazide moiety which expels N2 to form a nitrene which subsequently covalently labels the target nucleic acid. Structural analysis performed by high-resolution 2D NMR spectroscopy (400 MHz and 600 MHz) are reported for the model binary system 1:2:3, where 1 is the target 12-mer pdGTATCAGTTTCT, 2 is a photoactivatable fluoroazide derivative dAGAAACp-L-Az and 3 is the photosensitizer derivative Pyr-pdTGATAC (here: Az is the p-azidotetrafluorobenzyl group, Pyr the pyrenyl-1-methylamino group, L a linker group). The assignment of oligonucleotide and modifying group protons was performed using 1H COSY, TOCSY and NOESY experiments. Comprehensive analysis of 1H NOESY spectra of 1:2:3 showed that terminal fragments of the complex [5'p-1T-2G-3A-4T-], [-21A-22T-23A-24C], [-8T-9T-10T-11C-12T] and [13A-14G-15A-15A-17A-18C-] gave a continuous set of intra- and inter-nucleotide interactions, typical of regular double-stranded B-DNA. In contrast, the central region of the complex composed of 5C, 6A, 7G, 19T and 20G nucleotide residues, nearest the Pyr and Az groups, was found to be distorted. Thus some signals from aromatic and/or sugar-ring protons of the above nucleotide residues were extremely broadened or almost absent. Moreover, some intra- and/or inter-nucleotide interactions, typical of the regular DNA duplex, were not detected for the [-5C-6A-7G-] and [-19T-20G-] regions of the tandem system. Instead of that, some cross-peaks of low-intensity between the H2 proton of the Pyr group and 7G(H1'), 7G(H2'/H2"), 7G(H3'), 4T(H2"), 4T(H4') and 4T(H5'/H5") were observed. Additional 1H -1H NOE-interactions between methylene protons of the linker group L and some sugar ring protons of 18C nucleotide residue were detected. A preliminary structural model, constructed using proton-proton distances between Pyr and the DNA and Az-L and DNA obtained from a 1H NOESY experiment at 300 ms mixing time as constraints for the refinement of the structure, displayed significant distortion from B-DNA of the double-stranded helix in the middle of the complex, (-5C-6A-7G, -18C-19T-20G-). The Pyr group was located in what remains of the minor groove near 4T, 5C, 6A and 7G and the centroid of the azide ring less than 9A degrees from the centroid of the ring system of Pyr group.     

Comparison of the solution structures of intramolecular DNA triple helices containing adjacent and non-adjacent CG.C+ triplets

The solution conformations of the intramolecular triple helices d(AGAAGA-X-TCTTCT-X-TC+TTC+T) and d(AAGGAA-X-TTCCTT-X-TTC+C+TT) (X = non-nucleotide linker) have been determined by NMR.1H NMR spectra in H2O showed that the third strand cytosine residues are fully paired with the guanine residues, each using two Hoogsteen hydrogen bonds. Determination of the13C chemical shifts of the cytosine C6 and C5 and their one-bond coupling constants (1 J CH) conclusively showed that the Hoogsteen cytosine residues are protonated at N3. The global conformations of the two molecules determined with >19 restraints per residue are very similar (RMSD = 0.96 A). However, some differences in local conformation and dynamics were observed for the central two base triplets of the two molecules. The C N3H were less labile in adjacent CG.C+triplets than in non-adjacent ones, indicating that the adjacent charge does not kinetically destabilize these triplets. The sugar conformations of the two adjacent cytosine residues were different and the 5'-residue was atypical of protonated cytosine. Hence, there are subtle effects of the interaction between two adjacent cytosine residues. The central two purines in each sequence showed non-standard backbone conformations, averaging between gamma approximately 60 degrees and gamma approximately 180 degrees. This may be related to the difference in the dependence of the thermodynamic stability on pH observed for these two sequences.     

Investigation of substrate activation by 4-chlorobenzoyl-coenzyme A dehalogenase

4-Chlorobenzoyl-coenzyme A (4-CBA-CoA) dehalogenase catalyzes the hydrolysis of 4-CBA-CoA to 4-hydroxybenzoyl-coenzyme A (4-HBA-CoA), using the carboxylate side chain of aspartate 145 to displace the chloride from C(4) of the benzoyl ring. Previous UV-visible, Raman, and 13C NMR studies of enzyme-bound substrate analog or product ligand indicated that the environment of the enzyme active site induces a significant reorganization of the benzoyl ring pi-electrons. This observation was interpreted as evidence for electrophilic catalysis [viz. active-site-induced polarization of electron density away from the ring C(4)] [Taylor, K. L., Liu, R.-Q., Liang, P.-H., Price, J., Dunaway-Mariano, D., Tonge, P. J., Clarkson, J., & Carey, P. R. (1995) Biochemistry 34, 13881]. The recent crystal structure of the dehalogenase-4-HBA-CoA complex reveals two hydrogen bonds contributed to the benzoyl C=O by the backbone amide protons of Gly114 and Phe64 and a possible dipolar interaction with the positive pole of the 114-121 alpha-helix. Residues closely surrounding the benzoyl ring include W137, D145, W89, F64, F82, and H90. In the present study, the mutants D145A, H90Q, W137F, W89F, W89Y, F64L, F82L, and G114A were prepared to examine the effect of amino acid substitution on catalysis and on perturbation of the UV-visible spectral properties of the substrate benzoyl ring. Substitution of the two catalytic residues D145 and H90 inhibited catalysis but not ligand binding or the induction of the red shift in the benzoyl ring absorption. These two residues do not appear to contribute to substrate benzoyl ring binding or polarization. The F64L, F82L, W89F, and W137F mutants retained substantial catalytic activity and the ability to induce the red shift. The W89Y mutant, on the other hand, is inhibited in catalysis and ligand binding, suggesting that hydrophobicity more than packing may be critical for the benzoyl ring binding/activation. The G114A mutant was shown to be strongly inhibited in both substrate binding and activation, indicating that H-bonding and/or interaction with the dipole of the 114-121 alpha-helix may be crucial.     

13C NMR, X-ray, and differential scanning calorimetry investigations of truncated BPTI (aprotinin) analogues

Truncated BPTI missing residues 1 and 2 is investigated together with variants thereof (Lys-15, Arg-17, and Arg-42 are replaced by other residues in various combinations). A comparison of the X-ray structure of BPTI with that of 3-58BPTI(K15R,R17A,R42S) shows only minor variations for the backbone, but the lack of salt bridge between the terminals and the lack of two N-terminal residues provide a structure open at one end. Comparisons of amide exchange rates show a dramatic increase for the most slowly exchanging NH protons of 3-58BPTI and the analogues thereof, as compared to those of the wild-type despite only small differences in the structures. The amide exchange rates for truncated analogues increase with decreasing TTEP (temperature top endothermic peak) values. On the basis of the known structural changes comparisons to 13C chemical shifts are made. 13C chemical shifts are assigned using the D-isotope and HMBC techniques. Excellent resolution is obtained in these 1D natural abundance spectra. 13C NMR chemical shifts are shown to be able to gauge structural changes. A comparison of 13C chemical shifts of WT BPTI (aprotinin) and 3-58BPTI reveals effects caused by (i) the removal of the salt bridge of the terminii, (ii) the charge of the N-terminus, and (iii) the increased mobility of the side chain of Tyr-23. Small effects are also seen due to a conformational change of the aromatic ring of Phe-4. Ring current shifts at 13C chemical shifts are calculated. The difference in the calculated ring current effects are small comparing the wild-type with 3-58BPTI(K15R,R17A,R42S) provided the structures are relaxed. Protein unfolding as a function of pH and temperature is studied by DSC. Unfolding occurs at lower temperature with N-terminally truncated analogues, and the maximum is shifted toward higher pH.     

Structural characterization of an N-acetyl-2-aminofluorene (AAF) modified DNA oligomer by NMR, energy minimization, and molecular dynamics

An N-acetyl-2-aminofluorene (AAF) modified deoxyoligonucleotide duplex, d(C1-C2-A3-C4-[AAF-G5]-C6-A7-C8-C9).d(G10-G11-T12-G13-C14-++ +G15-T16-G17-G18), was studied by one- and two-dimensional NMR spectroscopy. Eight of the nine complementary nucleotides form Watson-Crick base pairs, as shown by NOEs between the guanine imino proton and cytosine amino protons for G.C base pairs or by an NOE between the thymine imino proton and adenine H2 proton for A.T base pairs. The AAF-G5 and C14 bases show no evidence of complementary hydrogen bond formation to each other. The AAF-G5 base adopts a syn conformation, as indicated by NOEs between the G5 imino proton and the A3-H3' and A3-H2'/H2" protons and by NOEs between the fluorene-H1 proton of AAF and the G5-H1' or C6-H1' proton. The NOEs from the C4-H6 proton to C4 sugar protons are weak, and thus the glycosidic torsion angle in this nucleotide is not well defined by these NMR data. The remaining bases are in the anti conformation, as depicted by the relative magnitude of the H8/H6 to H2' NOEs when compared to the H8/H6 to H1' NOEs. The three base pairs on each end of the duplex exhibit NOEs characteristic of right-handed B-form DNA. Distance restraints obtained from NOESY data recorded at 32 degrees C using a 100-ms mixing time were used in conformational searches by molecular mechanics energy minimization studies. The final, unrestrained, minimum-energy conformation was then used as input for an unrestrained molecular dynamics simulation. Chemical exchange cross peaks are observed, and thus the AAF-9-mer exists in more than a single conformation on the NMR time scale. The NMR data, however, indicate the presence of a predominant conformation (> or = 70%). The structure of the predominant conformation of the AAF-9-mer shows stacking of the fluorene moiety on an adjacent base pair, exhibiting features of the base-displacement [Grunberger, D., Nelson, J. H., et al. (1970) Proc. Natl. Acad. Sci. U.S.A. 66, 488-494] and insertion-denaturation models [Fuchs, R.P.P., & Daune, M. (1971) FEBS Lett. 14, 206-208], while the distal ring of the fluorene moiety protrudes into the minor groove.     

H NMR probes for inter-segmental hydrogen bonds in myoglobins

NMR signals arising from the HisB5 N delta H and HisEF5 N epsilon H protons in sperm whale skeletal and horse heart myoglobins have been located for the first time in the downfield shifted portion of the spectra. The shifts and hydrogen exchange rates indicate that these His imidazole ring NH protons are involved in the inter-segmental hydrogen bonds of the protein in solution, as demonstrated by a crystallographic study [Takano, T. (1977) J. Mol. Biol. 220, 381-399]. The assigned His imidazole ring NH proton resonances can serve as new sensitive structural probes in the study of the local conformation of myoglobin. The applicability of the NMR spectral parameters in the study of the tertiary structure of apomyoglobin, the denaturation of the protein, and the protein stability of sperm whale and horse myoglobins is presented in some detail.     

Three-dimensional structure of the ion-channel forming peptide trichorzianin TA VII bound to sodium dodecyl sulfate micelles

Trichorzianin TA VII, Ac0 U1 A2 A3 U4 J5 Q6 U7 U8 U9 S10 L11 U12 P13 V14 U15 I16 Q17 Q18 Fol19, is a nonadecapeptide member of the peptaibol antibiotics biosynthesized by Trichoderma soil fungi, which is characterized by a high proportion of the alpha, alpha-dialkylated amino acids, alpha-aminoisobutyric acid (Aib, U) and isovaline (Iva, J), an acetylated N-terminus and a C-terminal phenylalaninol (Pheol, Fol). The main interest in such peptides stems from their ability to interact with phospholipid bilayers and form voltage-dependent transmembrane channels in planar lipid bilayers. In order to provide insights into the lipid-peptide interaction promoting the voltage gating, the conformational study of TA VII in the presence of perdeuterated sodium dodecyl sulfate (SDS-d25) micelles has been carried out. 1H sequential assignment have been performed with the use of two-dimensional homo- and -heteronuclear nmr techniques including double quantum filtered correlated spectroscopy, homonuclear Hartmann-Hahn, nuclear Overhauser effect spectroscopy, 1H-13C heteronuclear single quantum correlation, and heteronuclear multiple bond correlation. Conformational parameters, such as 3JNHC alpha H coupling constants, temperature coefficients of amide protons (delta gamma/delta TNH) and quantitative nuclear Overhauser enhancement data, lead to detailed structural information. Ninety-eight three-dimensional structures consistent with the nmr data were generated from 231 interproton distances six phi dihedral angle restraints, using restrained molecular dynamics and energy minimization calculations. The average rms deviation between the 98 refined structures and the energy-minimized average structure is 0.59 A for the backbone atoms. The structure of trichorzianin TA VII associated with SDS micelles, as determined by these methods, is characterized by two right-handed helical segments involving residues 1-8 and 11-19, linked by a beta-turn that leads to an angle about 90 degrees-100 degrees between the two helix axes; residues 18 and 19 at the end of the C-terminal helix exhibit multiple conformations.     

Effect of phospholipase C on lymphocyte responses to mitogen

A reinvestigation of flavin phosphate synthesis, separation, identification, and interconversion was made in view of contradictory results in the literature. It has been confirmed that monochlorophosphoric acid is the best agent for selective 5'-monophosphorylation of riboflavin and derivatives. This reaction yields, however, invariably up to 20% of an isomer, which has been separated by preparative thick-layer chromatography and shown to be the 4'-monophosphate. All the earlier authors failed to detect this isomer which does not bind to flavodoxin. It equilibrates in dilute mineral acid to yield an 8:2 mixture of 5'-phosphate to 4'-phosphate by phosphate migration. The formation of 2',3',4'-triacetyl-flavin mononucleotide, according to Christie, S. M. H., Kenner, G. W. & Todd, A. R. (1954) J. Chem. Soc., 46-52, upon acid-catalysed acetylation of pure FMN, was confirmed. The same reaction under base catalysis, however, does not yield 2',3',4'-triacetyl-flavin mononucleotide as claimed by Khomutova, E. D., Shapiro, T. A., Mezentseva, M. W. & Berezovskii, V. M. (1965) Otd. Obshch. i. Tekhn. Khim., 241-244, Chem. Abstr. 65, 5516a, but in fact up to 80% 2', 3'-diacetyl-flavin 4':5'-cyclophosphate as the main product, which is stable under neutral and weak acidic conditions and does not hydrolyse to 2',3'-diacetyl-flavin 5'-monophosphate as claimed by McCormick, D.B. (1974) J. Heterocycl. Chem. 11, 969-974. The various flavin phosphates and their acetyl derivatives have been identified by pH-titration, electrophoresis, and proton magnetic resonance spectrometry, which direct analyses of crude reaction products as well as a rapid purity check of commerical FMN.     

(13C)-substituted sucrose: 13C-1H and 13C-13C spin coupling constants to assess furanose ring and glycosidic bond conformations in aqueous solution

Sucrose (beta-D-fructofuranosyl alpha-D-glucopyranoside, 1), methyl alpha-D-fructofuranoside (2), and methyl beta-D-fructofuranoside (3) have been prepared by chemical and/or enzymic methods with single sites of 13C-substitution at C-1, C-2, C-3, and C-6 of the fructofuranosyl ring. 1H (500 MHz) and 13C (75 and 125 MHz) NMR spectra of 1-3 have been obtained, yielding 1H-1H, 13C-1H, and 13C-13C spin coupling constants that were used to assess furanose ring and glycoside bond conformations in aqueous (2H2O) solution. Results show that the conformational mobility of the furanosyl ring in 3 is altered when incorporated into 1. Furthermore, 13C-13C and 13C-1H spin couplings across the glycosidic linkage suggest a psi torsion angle different from that observed in the crystal (phi appears similar). Interplay between the strength of the exoanomeric effect and hydrogen bonding in solution may be responsible, in part, for the apparent conformational flexibility of 1. In addition, spin couplings in 2 and 3 have been compared to those measured previously in alpha-D-threo-pentulofuranose (4) and beta-D-threo-pentulofuranose (5), respectively, as a means to study the effect of glycosidation and hydroxymethyl substitution on the solution conformation of the 2-ketofuranose ring. The conversion of 4 to 2 is accompanied by minimal conformational change, whereas a significant change accompanies the conversion of 5 to 3, showing that the effect of substitution on ring conformation depends highly on ring configuration before and after substitution.     

Mechanism of phosphatidylinositol-specific phospholipase C: a unified view of the mechanism of catalysis

The mechanism of phosphatidylinositol-specific phospholipase C (PI-PLC) has been suggested to resemble that of ribonuclease A. The goal of this work is to rigorously evaluate the mechanism of PI-PLC from Bacillus thuringiensis by examining the functional and structural roles of His-32 and His-82, along with the two nearby residues Asp-274 and Asp-33 (which form a hydrogen bond with His-32 and His-82, respectively), using site-directed mutagenesis. In all, twelve mutants were constructed, which, except D274E, showed little structural perturbation on the basis of 1D NMR and 2D NOESY analyses. The H32A, H32N, H32Q, H82A, H82N, H82Q, H82D, and D274A mutants showed a 10(4)-10(5)-fold decrease in specific activity toward phosphatidylinositol; the D274N, D33A, and D33N mutants retained 0. 1-1% activity, whereas the D274E mutant retained 13% activity. Steady-state kinetic analysis of mutants using (2R)-1, 2-dipalmitoyloxypropane-3-(thiophospho-1d-myo-inositol) (DPsPI) as a substrate generally agreed well with the specific activity toward phosphatidylinositol. The results suggest a mechanism in which His-32 functions as a general base to abstract the proton from 2-OH and facilitates the attack of the deprotonated 2-oxygen on the phosphorus atom. This general base function is augmented by the carboxylate group of Asp-274 which forms a diad with His-32. The H82A and D33A mutants showed an unusually high activity with substrates featuring low pKa leaving groups, such as DPsPI and p-nitrophenyl inositol phosphate (NPIPs). These results suggest that His-82 functions as the general acid with assistance from Asp-33, facilitating the departure of the leaving group by protonation of the glycerol O3 oxygen. The Bronsted coefficients obtained for the WT and the D33N mutant indicate a high degree of proton transfer to the leaving group and further underscore the "helper" function of Asp-33. The complete mechanism also includes activation of the phosphate group toward nucleophilic attack by a hydrogen bond between Arg-69 and a nonbridging oxygen atom. The overall mechanism can be described as "complex" general acid-general base since three elements are required for efficient catalysis.     

U73122 and U73343 inhibit receptor-mediated phospholipase D activation downstream of phospholipase C in CHO cells

We investigated the effects of nitric oxide (NO) donors, S-nitroso-N-acetylpenicillamine and sodium nitroprusside on basal and K+-evoked release of [3H]noradrenaline from superfused synaptosomes from the rat cerebral cortex. Both substances produced concentration-dependent increases in the release of the labeled transmitter under basal and depolarized conditions. The effects of the donors on basal release were Ca2+-independent but were not inhibited by the carrier-uptake blocker, desipramine; the effects were abolished by hemoglobin (an NO scavenger). Thirty-five minutes after stimulation with sodium nitroprusside, the synaptosomes were still responsive to KCl stimulation, indicating that the donor's effects were not caused by damage to the synaptosome membrane. The cGMP analogue, 8-bromo-cGMP, had no effect on basal release, and the enhanced release produced by sodium nitroprusside was not inhibited by the specific inhibitor of soluble guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one, indicating that NO's effects on basal release of the neurotransmitter are guanylate cyclase-independent. Both of the NO donors had more marked effects on release of [3H]noradrenaline during K+-stimulated depolarization. The NO-mediated increase in this case was partially antagonized by 10 microM LH-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one, and 8-Br-cGMP was also capable of producing concentration-dependent increases in the K+-stimulated release of the transmitter. These findings indicate that the effects of the NO donors on [3H]noradrenaline release during depolarization are partially mediated by the activation of guanylate cyclase.     

Structure of gene 5 protein-oligodeoxynucleotide complexes as determined by 1H, 19F, and 31P nuclear magnetic resonance

1H nuclear magnetic resonance (NMR) spectra at 270 MHz of gene 5 protein from bacteriophage fd and its complexes with tetra- and octadeoxynucleotides show that approximately 12 of the 37 aromatic protons of the protein undergo upfield shifts upon nucleotide binding. In the complex with d(pT)8, the upfield shifts of the aromatic protons average approximately 0.3 ppm, while in the d(pA)8 complex the same resonances (assigned to tyrosyl protons) shift upfield approximately 0.8 ppm. These are interpreted as ring current shifts induced by stacking of the phenyl rings of three of the five tyrosyl residues with the bases of the nucleotides. 19FNMR of m-fluorotyrosyl gene 5 protein shows five separate resonances: two downfield from m-fluorotyrosine corresponding to "buried" tyrosyls and three near m-fluorotyrosine corresponding to "surface" tyrosyls. The latter (assigned to Tyr-26, -41, and -56, shown by chemical modification to be exposed to solvent) move upfield on nucleotide binding. The downfield 19F resonances are unaffected. Thus the aromatic protons shifted upfield on nucleotide binding appear to be those of Tyr-26, -41, and -56. In contrast to tetra-, octanucleotide binding to gene 5 protein induces large changes in the 1H resonances of the -CH3 groups of the Val, Leu, and Ile side chains. These may reflect conformational changes induced by protein-protein interactions between two monomers bound to the octanucleotide. 1H resonances of the epsilon-CH2 groups of the lysyl residues in the protein and the complexes with nucleotides are narrow with long T2 values, suggesting considerable rotational motion. Thus epilson-NH3+-phosphate interactions, if they occur, are on the surface of the complex and allow the epsilon-CH2 groups to retain considerable rotational freedom. 31P NMR of the bound nucleotides shows large decreases in T1 for the 3'-5' diesters, but little chemical shift suggesting no unusual distortion of the nucleotide backbone on binding to gene 5 protein. A three-dimensional model of a gene 5 protein-octanucleotide complex has been built based on predictions of the secondary structure from the amino acid sequence (87 AA) and tertiary folding dictated by known chemical and NMR features of the complex.     

Long-range effects on dynamics in a temperature-sensitive mutant of trp repressor

A mutant tryptophan repressor (TrpR) protein containing the substitution of phenylalanine for leucine 75 has been isolated following a genetic screen for temperature-sensitive mutations. Two-dimensional (2D) 1H NMR spectra indicate an overall very similar fold for the purified mutant and wild-type proteins. Circular dichroism spectropolarimetry indicates an increased helix content relative to the wild-type protein, and a slightly higher urea denaturation midpoint for the mutant protein, although there is no difference in thermal stability. Fluorescence spectra indicate a more buried environment for one or both tryptophan residues in the mutant protein. The rate of proton-deuterium exchange-out for the resolved indole ring protons of the two tryptophan residues was quantified from NMR spectra of mutant and wild-type proteins and found to be approximately 50% faster in the wild-type protein. The mutant protein binds the corepressor l-tryptophan (l-Trp) approximately ten times more weakly than does the wild-type protein, but in l-Trp excess its DNA-binding affinity is only two to fivefold weaker. Taken together the results imply that, despite its conservative chemical character and surface location at the C terminus of helix one in the helix-turn-helix DNA recognition motif, this mutational change confers long-range effects on the dynamics of the protein's secondary and tertiary structure without substantially altering its fold, and with relatively minor effects on protein function.     

Probing the roles of active site residues in phosphatidylinositol-specific phospholipase C from Bacillus cereus by site-directed mutagenesis

The role of amino acid residues located in the active site pocket of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus[Heinz, D. W., Ryan, M., Bullock, T., & Griffith, O. H. (1995) EMBO J. 14, 3855-3863] was investigated by site-directed mutagenesis, kinetics, and crystal structure analysis. Twelve residues involved in catalysis and substrate binding (His32, Arg69, His82, Gly83, Lys115, Glu117, Arg163, Trp178, Asp180, Asp198, Tyr200, and Asp274) were individually replaced by 1-3 other amino acids, resulting in a total number of 21 mutants. Replacements in the mutants H32A, H32L, R69A, R69E, R69K, H82A, H82L, E117K, R163I, D198A, D198E, D198S, Y200S, and D274S caused essentially complete inactivation of the enzyme. The remaining mutants (G83S, K115E, R163K, W178Y, D180S, Y200F, and D274N) exhibited reduced activities up to 57% when compared with wild-type PI-PLC. Crystal structures determined at a resolution ranging from 2.0 to 2.7 A for six mutants (H32A, H32L, R163K, D198E, D274N, and D274S) showed that significant changes were confined to the site of the respective mutation without perturbation of the rest of the structure. Only in mutant D198E do the side chains of two neighboring arginine residues move across the inositol binding pocket toward the newly introduced glutamic acid. An analysis of these structure-function relationships provides new insight into the catalytic mechanism, and suggests a molecular explanation of some of the substrate stereospecificity and inhibitor binding data available for this enzyme.     

Solution structure of the oxidized 2[4Fe-4S] ferredoxin from Clostridium pasteurianum

Following the recently developed approach to the solution structure of paramagnetic high-potential iron-sulfur proteins, the three-dimensional structure in solution of the oxidized Clostridium pasteurianum ferredoxin has been solved by 1H-NMR. The X-ray structure is not available. The protein contains 55 amino acids and two [4Fe-4S] clusters. In the oxidized state, the clusters have S = 0 ground states, but are paramagnetic because of thermal population of excited states. Due to the somewhat small size of the protein and to the presence of two clusters, approximately 55% of the residues have at least one proton with a non-selective T1 smaller than 25 ms. The protein has thus been used as a test system to challenge the present paramagnetic NMR methodology both in achieving an extended assignment and in obtaining a suitable number of constraints. 79% of protein protons have been assigned. Analogy with other ferredoxins of known structure has been of help to speed up the final stages of the assignment, although we have shown that this independent information is not necessary. In addition to dipolar connectivities, partially detected through tailored experiments, 3JHN-H alpha, H-bond constraints and dihedral angle constraints on the Cys chi 2 angles have been generated by using a recently derived Karplus-type relationship for the hyperfine shifts of cysteine beta CH2 protons. In total, 456 constraints have been used in distance geometry calculations. The final quality of the structures is satisfactory, with root-mean-square deviation values of 66 pm and 108 pm for backbone and heavy atoms, respectively. The resulting structure is compared with that of Clostridium acidi urici ferredoxin [Duée, E. D., Fanchon, E., Vicat, J., Sieker, L. C., Meyer, J. & Moulis, J.-M. (1994) J. Mol. Biol. 243, 683-695]. The two proteins are very similar in the overall folding, secondary structure elements and side-chain orientations. The C alpha root-mean-square deviation values between the X-ray-determined C. acidi urici ferredoxin structure and the conformer with lowest energy of the C. pasteurianum ferredoxin family is 78 pm (residues 3-53). Discrepancies in residues 26-28 may arise from the disorder observed in the X-ray structure in that region.     

Biosynthesis of pteridines. NMR studies on the reaction mechanisms of GTP cyclohydrolase I, pyruvoyltetrahydropterin synthase, and sepiapterin reductase

GTP cyclohydrolase I catalyzes a ring expansion affording dihydroneopterin triphosphate from GTP. [1',2',3',4',5'-13C5, 2'-2H1]GTP was prepared enzymatically from [U-13C6]glucose for use as enzyme substrate. Multinuclear NMR experiments showed that the reaction catalyzed by GTP cyclohydrolase I involves the release of a proton from C-2' of GTP that is exchanged with the bulk solvent. Subsequently, a proton is reintroduced stereospecifically from the bulk solvent. This is in line with an Amadori rearrangement mechanism. The proton introduced from solvent occupies the pro-7R position in the enzyme product. The data also confirm that the reaction catalyzed by pyruvoyltetrahydropterin synthase results in the incorporation of solvent protons into positions C-6 and C-3' of the enzyme product. On the other hand, the reaction catalyzed by sepiapterin reductase does not involve any detectable incorporation of solvent protons into tetrahydrobiopterin.     

A heterologous system for detecting eukaryotic enzymes which synthesize pseudouridine in transfer ribonucleic acids

tRNA pseudouridylation activities have been detected in embryonic mouse cell fractions and in extracts from HeLa, mouse L-cell and baby hamster kidney (BHK) cell lines. These activities were identified by the use of heterologous reaction systems, with tRNA from hisT strains of Salmonella typhimurium as substrate. hisT mutants are defective for an enzyme that forms psi residues in the anticodon region of many tRNAs and accumulate undermodified species of tRNA. The pseudouridylation activity from BHK cells has been examined in detail and quantitated by a modified tritium release assay (Cortese, R., Kammen, H.O., Spengler, S.J., and Ames, B.N. (1974) J. Biol. Chem. 249, 1103-1108). Maximal rates of tritium release required a suitable cationic environment (optimally, a combination of Mg2+ and NH4+) and a thiol reductant. The activity was totally inhibited in the presence of thiol-reactive reagents, such as 5,5'-dithiobis(2-nitrobenzoic acid) and p-chloromercuribenzoate. A major portion of this 3H release activity was associated with psi modification reactions. This conclusion stems from the following observations: (a) BHK extracts preferentially catalyzed a release of 3H from hisT [5-3H]tRNA, rather than from similarly labeled wild type tRNA; (b) this activity was specific for protons attached to C5 of the pyrimidine rings; no release of 3H was obtained with hisT or wild type [6-3H]tRNA as substrate; (c) the reaction products of hisT tRNA with BHK enzyme were examined by reverse phase column chromatography of tRNAPhe isoacceptors on RPC-5 columns. The enzyme modified both of the principal isoacceptors of hisT tRNAPhe to an equal extent, yielding products indistinguishable from wild type tRNAPhe. Significant levels of 3H release were obtained by the action of enzyme on wild type [5-3H]tRNA, even after gel filtration of the enzyme. This suggests that the enzyme may be able to hypermodify certain species of wild type S. typhimurium tRNA. The activities for wild type tRNA and hisT tRNA appeared to be associated with the same enzyme.     

Enzymes of carbohydrate metabolism in naevoid basal-cell epithelioma

The methods which have been used for the observation and assignment of resonances in the NMR spectra of proteins are reviewed. One such method, the selective deuteration of the aromatic protons of tryptophyl residues, is studied by NMR spectroscopy in model compounds in this paper, and in proteins in the following paper. On the basis of a reassignment of the PMR spectrum of the aromatic protons of L-tryptophan, the relative rates of H-D exchange in deutero-trifluoracetic acid (d-TFA) are H-2 greater than H-5 greater than H-6 greater than H-4 approximately H-7. The energies of activation for the first order exchange of both the H-2 and H-5 protons is 12 k.cal.mol-1. The rate constant for exchange of the H-2 protons of tryptophyl residues in peptides is much greater than in the amino acid itself and 5-10 times that for exchange of the H-5 protons. This suggests that the method can be used to label tryptophyl residues in proteins rapidly and specifically.