Thermolysin-inhibitor complexes examined by 31P and 113Cd NMR spectroscopy

Complexes between phosphoramidon (N-(alpha-rhamnopyranosyloxyhydroxyphosphinyl)-L-leucyl-L-tryptoph an) and zinc thermolysin and between phosphoramidon or N-phosphoryl-L-leucineamide and 113Cd-substituted thermolysin have been examined by 31P and 113Cd NMR spectroscopy. 113Cd resonances are observed at 168 and 152 ppm for the phosphoramidon and N-phosphoryl-L-leucineamide complexes, respectively. There are large but different chemical shift anisotropy contributions to the 113Cd line widths for the two complexes, which reflect the known structural differences for the zinc-enzyme complexes. 113Cd-31P spin-spin coupling is also seen and differs for the two cadmium complexes, being larger, 28 Hz, for the bidentate N-phosphoryl-L-leucineamide ligand than for the monodentate phosphoramidon, 16 Hz. Large changes in chemical shift, 7.5-10.9 ppm, are seen for the 31P resonances of the inhibitors upon binding to the enzyme reflecting direct phosphoryl-metal ligation. Chemical shift anisotropy is the dominant relaxation mechanism for the 31P nuclei at 9.4 T, while the dipole-dipole contribution seems to be unaffected by a change of solvent from H2O to D2O.     

Active site contacts in the purine nucleoside phosphorylase--hypoxanthine complex by NMR and ab initio calculations

Hypoxanthine (Hx) with specific (15)N labels has been used to probe hydrogen-bonding interactions with purine nucleoside phosphorylase (PNP) by NMR spectroscopy. Hx binds to human PNP as the N-7H tautomer, and the N-7H (1)H and (15)N chemical shifts are located at 13.9 and 156.5 ppm, respectively, similar to the solution values. In contrast, the (1)H and (15)N chemical shifts of N-1H in the PNP.Hx complex are shifted downfield by 3.5 and 7.5 ppm to 15.9 and 178.8 ppm, respectively, upon binding. Thus, hydrogen bonding at N-1H is stronger than at N-7H in the complex. Ab initio chemical shift calculations on model systems that simulate Hx in solution and bound to PNP are used to interpret the NMR data. The experimental N-7H chemical shift changes are caused by competing effects of two active site contacts. Hydrogen bonding of Glu201 to N-1H causes upfield shifts of the N-7H group, while the local hydrogen bond (C=O to N-7H from Asn243) causes downfield shifts. The observed N-7H chemical shift can be reproduced by a hydrogen bond distance approximately 0.13 A shorter (but within experimental error) of the experimental value found in the X-ray crystal structure of the bovine PNP.Hx complex. The combined use of NMR and ab initio chemical shift computational analysis provides a novel approach to understand enzyme-ligand interactions in PNP, a target for anticancer agents. This approach has the potential to become a high-resolution tool for structural determination.     

31P NMR of covalent phosphorylated derivatives of alpha-chymotrypsin

The structures of various covalent phosphorylated derivatives of alpha-chymotrypsin (alpha-CT) have been studied by 31P NMR spectroscopy. Diisopropylphosphoryl-alpha-chymotrypsin (alpha-DIPCT) shows a single 31P signal at ca. 0.0 ppm (pH 4). At low pH, the 31P NMR spectrum of alpha-DIPCT gradually changed with the appearance of one or two additional peaks. The ratio of the peaks varied with pH, time, and concentration. One of these two new downfield peaks (both at ca. 2.0 ppm) has been previously identified by Markley and co-workers (Markley, 1979; Porubcan et al., 1979) and van der Drift et al. (1985) as an aged monoisopropylphosphoryl-alpha-chymotrypsin (alpha-MIPCT) and is confirmed by our studies. A new additional downfield signal, separate from the alpha-MIPCT signal, is attributed to a dimer of the phosphorylated alpha-DIPCT. Phosphorylation of the enzyme with diphenyl chlorophosphate yields a monophenylphosphoryl-alpha-chymotrypsin (alpha-MPPCT) that also showed a single 31P signal at -2.1 ppm (pH 7). However, the spectrum did not change as a function of pH, incubation time, or concentration. Comparison of the 31P chemical shifts of the native and denatured phosphorylated derivatives of alpha-chymotrypsin suggests changes in the conformation about the P-O ester bonds are at least partially responsible for the various 31P chemical shift differences.     

Mechanism of the binding of Z-L-tryptophan and Z-L-phenylalanine to thermolysin and stromelysin-1 in aqueous solutions

The chemical shift of the carboxylate carbon of Z-tryptophan is increased from 179.85 to 182.82 ppm and 182.87 ppm on binding to thermolysin and stromelysin-1 respectively. The chemical shift of Z-phenylalanine is also increased from 179.5 ppm to 182.9 ppm on binding to thermolysin. From pH studies we conclude that the pK(a) of the inhibitor carboxylate group is lowered by at least 1.5 pK(a) units when it binds to either enzyme. The signal at ~183 ppm is no longer observed when the active site zinc atom of thermolysin or stromelysin-1 is replaced by cobalt. We estimate that the distance of the carboxylate carbon of Z-[1-(13)C]-L-tryptophan is ≤3.71? from the active site cobalt atom of thermolysin. We conclude that the side chain of Z-[1-(13)C]-L-tryptophan is not bound in the S(2)' subsite of thermolysin. As the chemical shifts of the carboxylate carbons of the bound inhibitors are all ~183 ppm we conclude that they are all bound in a similar way most probably with the inhibitor carboxylate group directly coordinated to the active site zinc atom. Our spectrophotometric results confirm that the active site zinc atom is tetrahedrally coordinated when the inhibitors Z-tryptophan or Z-phenylalanine are bound to thermolysin.     

31P NMR spectra of ethidium, quinacrine, and daunomycin complexes with poly(adenylic acid).poly(uridylic acid) RNA duplex and calf thymus DNA

31P NMR provides a convenient monitor of the phosphate ester backbone conformational changes upon binding of the intercalating drugs ethidium, quinacrine, and daunomycin to sonicated poly(A).poly(U) and calf thymus DNA. 31P chemical shifts can also be used to assess differences in the duplex unwinding angles in the presence of the drug. Thus a new 31P signal, 1.8-2.2 ppm downfield from the double-stranded helix signals, is observed in the ethidium ion-poly(A).poly(U) complex. This signal arises from phosphates which are in perturbed environments due to intercalation of the drug. This is in keeping with the hypothesis that the P-O ester torsional angle in phosphates linking the intercalated base pairs is more trans-like. Similar though smaller deshielding of the 31P signals is observed in sonicated poly(A).poly(U)-quinacrine complexes as well as in the daunomycin complexes. The effect of added ethidium ion, quinacrine, and daunomycin on the 31P spectra of sonicated calf thymus DNA is consistent with Wilson and Jones' (1982) earlier study. In these drug-DNA complexes the drug produces a gradual downfield shift in the DNA 31P signal without the appearance of a separate downfield peak. These differences are attributed to differences in the rate of chemical exchange of the drug between free and bound duplex states. The previous correlation of 31P chemical shift with drug duplex unwinding angle (Wilson & Jones, 1982) is confirmed for both the RNA and DNA duplexes.     

31P NMR spectra of an oligodeoxyribonucleotide duplex lac operator-repressor headpiece complex

The interaction of a symmetric lac operator duplex, d(TGTGAGCGCTCACA)2, with the N-terminal 56-residue headpiece fragment of the lac repressor protein was monitored by 31P NMR spectroscopy. The changes in the 31P chemical shifts upon addition of the headpiece demonstrated an end point of two headpiece fragments per symmetric 14-mer duplex with each headpiece binding to the T1pG2pT3pG4pA5 ends of the duplex. The specific phosphate 31P perturbations observed are consistent with those residues implicated in protein binding by previous NMR, molecular biological, and biochemical techniques. Upon complexation, the 31P signals of phosphates G2-A5 showed upfield or downfield shifts (less than 0.2 ppm) while most other residues were unperturbed. The interactions were dependent on ionic strength. The 31P NMR data provide direct evidence for predominant recognition of the 5' strand of the 5'-TGTGA/3'-ACACT binding site.     

13C NMR analysis of electrostatic interactions between NAD+ and active site residues of UDP-galactose 4-epimerase: implications for the activation induced by uridine nucleotides

UDP-galactose 4-epimerase contains the coenzyme NAD+ bound tightly at the active site. NAD+ functions as the coenzyme for the interconversion of UDP-galactose and UDP-glucose by reversibly mediating their dehydrogenation to the common intermediate UDP-4-ketohexopyranoside. The epimerase structure and spectrophotometric data indicate that NAD+ may engage in electrostatic interactions with amino acid side chains that may regulate the reactivity of NAD+. In this work, we carried out NMR studies of [nicotinamide-4-13C]NAD+ bound to wild-type epimerase and epimerases mutated at amino acid residues in contact with NAD+. The 4-13C NMR chemical shifts revealed the following: The 4-13C chemical shift in wild-type epimerase is 149.9 ppm; mutation of Ser 124 to Ala changes it slightly by 0.2 ppm to 150.1 ppm; mutation of Tyr 149 to Phe results in a downfield perturbation of 2.7 ppm to 152.6 ppm; and the simultaneous mutation of Ser 124 to Ala and Tyr 149 to Phe also causes a downfield perturbation of 2.8 ppm to 152.7 ppm. Mutation of Lys 153 to Met results in a 13C chemical shift of 150.8 ppm, which is 0.9 ppm downfield from that of wild type and 1.8 ppm upfield from that of Y149F-epimerase. The 13C chemical shifts of nicotinamide C4 of NAD+ in these epimerases are correlated with their respective reactivities with NaBH3CN. In addition, reactivity of NAD+ in wild-type and S124A-epimerases displays pH dependence, with higher rates at lower pH where Tyr 149 in these two enzymes is protonated. The results support an electrostatic model in which repulsion between positively charged Lys 153 and N1 of the nicotinamide ring increases the reactivity of NAD+, while the phenolate of Tyr 149 opposes the positive electrostatic field and attenuates the reactivity of NAD+. Ser 124 has very little effect on the electron distribution within the nicotinamide ring or the reactivity of NAD+. The effects of binding the substrate analogue P1-uridyl-P2-methyl diphosphate (Me-UDP) on the 4-13C chemical shifts are opposite to those induced by the mutations. MeUDP perturbs the 4-13C chemical shift 2.9 ppm downfield in the wild-type and S124A-epimerases but has little or no effect in the cases of Y149F- or K153M-epimerases. The results support the postulate that NAD+ activation induced by uridine nucleotides is brought about by a conformational change of epimerase that repositions Tyr 149 at an increased distance from nicotinamide N1 of NAD+ while maintaining the electrostatic repulsion between Lys 153 and nicotinamide N1 of NAD+.     

Imino 1H- and 31P-NMR analysis of the interaction of propidium and ethidium with DNA

At low temperature and low salt concentration, both imino proton and ³¹p-nmr spectra of DNA complexes with the intercalators ethidium and propidium are in the slow-exchange region. Increasing temperature and/or increasing salt concentration results in an increase in the site exchange rate. Ring-current effects from the intercalated phenanthridinium ring of ethidium and propidium cause upfield shifts of the imino protons of A · T and G · C base pairs, which are quite similar for the two intercalators. The limiting induced chemical shifts for propidium and ethidium at saturation of DNA binding sites are approximately 0.9 ppm for A · T and 1.1 ppm for G · C base pairs. The similarity of the shifts for ethidium and propidium, in both the slow- and fast-exchange regions over the entire titration of DNA, shows that a binding model for propidium with neighbor-exclusion binding and negative ligand cooperativity is correct. The fact that a unique chemical shift is obtained for imino protons at intercalated sites over the entire titration and that no unshifted imino proton peaks remain at saturation binding of ethidium and propidium supports a neighbor-exclusion binding model with intercalators bound at alternating sites rather than in clusters on the double helix. Addition of ethidium and propidium to DNA results in downfield shifts in ³¹P-nmr spectra. At saturation ratios of intercalator to DNA base pairs in the titration, a downfield shoulder (approximately ?2.7 ppm) is apparent, which accounts for approximately 15% of the spectral area. The main peak is at ?3.9 to ?4.0 ppm relative to ?4.35 in uncomplexed DNA. The simplest neighbor-binding model predicts a downfield peak with approximately 50% of the spectral area and an upfield peak, near the chemical shift for uncomplexed DNA, with 50% of the area. This is definitely not the case with these intercalators. The observed chemical shifts and areas for the DNA complexes can be explained by models, for example, that involve spreading the intercalation-induced unwinding of the double helix over several base pairs and/or a DNA sequence- and conformation-dependent heterogeneity in intercalation-induced chemical shifts and resulting exchange rates.     

Observation of a short, strong hydrogen bond in the active site of hydroxynitrile lyase from Hevea brasiliensis explains a large pKa shift of the catalytic base induced by the reaction intermediate

The hydroxynitrile lyase from Hevea brasiliensis (HbHNL) uses a catalytic triad consisting of Ser(80)-His(235)-Asp(207) to enhance the basicity of Ser(80)-O gamma for abstracting a proton from the OH group of the substrate cyanohydrin. Following the observation of a relatively short distance between a carboxyl oxygen of Asp(207) and the N delta(1)(His(235)) in a 1.1 A crystal structure of HbHNL, we here show by (1)H and (15)N-NMR spectroscopy that a short, strong hydrogen bond (SSHB) is formed between the two residues upon binding of the competitive inhibitor thiocyanate to HbHNL: the proton resonance of H-N delta 1(His(235)) moves from 15.41 ppm in the free enzyme to 19.35 ppm in the complex, the largest downfield shift observed so far upon inhibitor binding. Simultaneously, the D/H fractionation factor decreases from 0.98 to 0.35. In the observable pH range, i.e. between pH 4 and 10, no significant changes in chemical shifts (and therefore hydrogen bond strength) were observed for free HbHNL. For the complex with thiocyanate, the 19.35 ppm signal returned to 15.41 ppm at approximately pH 8, which indicates a pK(a) near this value for the H-N epsilon(2)(His(235)). These NMR results were analyzed on the basis of finite difference Poisson-Boltzmann calculations, which yielded the relative free energies of four protonation states of the His(235)-Asp(207) pair in solution as well as in the protein environment with and without bound inhibitor. The calculations explain all the NMR features, i.e. they suggest why a short, strong hydrogen bond is formed upon inhibitor binding and why this short, strong hydrogen bond reverts back to a normal one at approximately pH 8. Importantly, the computations also yield a shift of the free energy of the anionic state relative to the zwitterionic reference state by about 10.6 kcal/mol, equivalent to a shift in the apparent pK(a) of His(235) from 2.5 to 10. This huge inhibitor-induced increase in basicity is a prerequisite for His(235) to act as general base in the HbHNL-catalyzed cyanohydrin reaction.     

15N-labeled tRNA. Identification of 4-thiouridine in Escherichia coli tRNASer1 and tRNATyr2 by 1H-15N two-dimensional NMR spectroscopy

Uridine is uniquely conserved at position 8 in elongator tRNAs and binds to A14 to form a reversed Hoogsteen base pair which folds the dihydrouridine loop back into the core of the L-shaped molecule. On the basis of 1H NMR studies, Hurd and co-workers (Hurd, R. E., Robillard, G. T., and Reid, B. R. (1977) Biochemistry 16, 2095-2100) concluded that the interaction between positions 8 and 14 is absent in Escherichia coli tRNAs with only 3 base pairs in the dihydrouridine stem. We have taken advantage of the unique 15N chemical shift of N3 in thiouridine to identify 1H and 15N resonances for the imino units of S4U8 and s4U9 in E. coli tRNASer1 and tRNATyr2. Model studies with chloroform-soluble derivatives of uridine and 4-thiouridine show that the chemical shifts of the protons in the imino moieties move downfield from 7.9 to 14.4 ppm and from 9.1 to 15.7 ppm, respectively; whereas, the corresponding 15N chemical shifts move downfield from 157.5 to 162.5 ppm and from 175.5 to 180.1 ppm upon hydrogen bonding to 5'-O-acetyl-2',3'-isopropylidene adenosine. The large difference in 15N chemical shifts for U and s4U allows one to unambiguously identify s4U imino resonances by 15N NMR spectroscopy. E. coli tRNASer1 and tRNATyr2 were selectively enriched with 15N at N3 of all uridines and modified uridines. Two-dimensional 1H-15N chemical shift correlation NMR spectroscopy revealed that both tRNAs have resonances with 1H and 15N chemical shifts characteristic of s4UA pairs. The 1H shift is approximately 1 ppm upfield from the typical s4U8 resonance at 14.8 ppm, presumably as a result of local diamagnetic anisotropies. An additional s4U resonance with 1H and 15N shifts typical of interaction of a bound water or a sugar hydroxyl group with s4U9 was discovered in the spectrum of tRNATyr2. Our NMR results for tRNAs with 3-base pair dihydrouridine stems suggest that these molecules have an U8A14 tertiary interaction similar to that found in tRNAs with 4-base pair dihydrouridine stems.     

Unique cyanide nitrogen-15 nuclear magnetic resonance chemical shift values for cyano-peroxidase complexes. Relevance to the heme active-site structure and mechanism of peroxide activation

Cyanide ion has been utilized to probe the heme environment of the ferric states of horseradish peroxidase, lactoperoxidase and chloroperoxidase. The 15N-NMR signal for cyanide bound to these enzymes is located in the downfield region from 578 to 412 ppm (with respect to the nitrate ion reference). The corresponding signal for met-forms of hemoglobin, myoglobin and cytochrome c is much further downfield in the 1047-847 ppm region. The signal position for peroxidases is quite invariant with pH in the physiological ranges. The upfield bias for peroxidase chemical shifts must reflect unique trans iron(III) ligand types and/or proximal-group hydrogen bonding or steric effects. Model compound studies reveal a significant upfield cyanide 15N shift with addition of agents capable of hydrogen-bonding to the coordinated cyanide ion. An even more striking upfield shift of 277 ppm is associated with deprotonation of a trans imidazole residue. The distinctive chemical shifts observed for the cyano ligand in peroxidases support the hypothesis that a distal hydrogen-bonding network and perhaps a polar, basic trans ligand are essential for O-O bond activation by peroxidases.     

Nuclear magnetic resonance study of the Schiff base in bacteriorhodopsin: counterion effects on the 15N shift anisotropy

High-resolution, solid-state 15N NMR has been used to study the chemical shift anisotropies of the Schiff bases in bacteriorhodopsin (bR) and in an extensive series of model compounds. Using slow-spinning techniques, we are able to obtain sufficient rotational sideband intensity to determine the full 15N chemical shift anisotropy for the Schiff base nitrogen in bR548 and bR568. Comparisons are made between all-trans-bR568 and N-all-trans-retinylidene butylimine salts with halide, phenolate, and carboxylate counterions. It is argued that for the model compounds the variation in 15N chemical shift reflects the variation in (hydrogen) bond strength with the various counterions. The results suggest that carboxylates and tyrosinates may form hydrogen bonds of comparable strength in a hydrophobic environment. Thus, the hydrogen bonding strength of a counterion depends on factors that are not completely reflected in the solution pKa of its conjugate acid. For the model compounds, the two most downfield principal values of the 15N chemical shift tensor, sigma 22 and sigma 33, vary dramatically with different counterions, whereas sigma 11 remains essentially unaffected. In addition, there exists a linear correlation between sigma 22 and sigma 33, which suggests that a single mechanism is responsible for the variation in chemical shifts present in all three classes of model compounds. The data for bR568 follow this trend, but the isotropic shift is 11 ppm further upfield than any of the model compounds. This extreme value suggests an unusually weak hydrogen bond in the protein.     

Mechanism of the binding of Z-L-tryptophan and Z-L-phenylalanine to thermolysin and stromelysin-1 in aqueous solutions

The chemical shift of the carboxylate carbon of Z-tryptophan is increased from 179.85 to 182.82 ppm and 182.87 ppm on binding to thermolysin and stromelysin-1 respectively. The chemical shift of Z-phenylalanine is also increased from 179.5 ppm to 182.9 ppm on binding to thermolysin. From pH studies we conclude that the pK_a of the inhibitor carboxylate group is lowered by at least 1.5 pK_a units when it binds to either enzyme. The signal at ~ 183 ppm is no longer observed when the active site zinc atom of thermolysin or stromelysin-1 is replaced by cobalt. We estimate that the distance of the carboxylate carbon of Z-[1-¹³C]-L-tryptophan is ≤ 3.71 Å from the active site cobalt atom of thermolysin. We conclude that the side chain of Z-[1-¹³C]-L-tryptophan is not bound in the S₂′ subsite of thermolysin. As the chemical shifts of the carboxylate carbons of the bound inhibitors are all ~ 183 ppm we conclude that they are all bound in a similar way most probably with the inhibitor carboxylate group directly coordinated to the active site zinc atom. Our spectrophotometric results confirm that the active site zinc atom is tetrahedrally coordinated when the inhibitors Z-tryptophan or Z-phenylalanine are bound to thermolysin.     

(13)C, (31)P and (15)N NMR studies of the ligand exchange reactions of auranofin and chloro(triethylphosphine)gold(I) with thiourea.

The interaction of thiourea (Tu) with auranofin (Et(3)PAuSATg) and its analogue, Et(3)PAuCl has been studied using (13)C, (31)P and (15)N NMR spectroscopy. It is observed that Tu is able to replace both the ligands, Et(3)P and SATg(-) simultaneously from gold(I) in auranofin, forming [Et(3)P-Au-Tu](+) and Tu-Au-SATg complexes. However, no separate resonances for these species were observed either due to their rapid exchange with auranofin and thus giving only the average resonances or because the chemical shifts of either two species are same so that they cannot be resolved. The displaced SATg(-) is oxidized to its disulfide, (SATg)(2). However, some of the displaced Et(3)P is oxidized to Et(3)PO while the remaining reacts with Tu to form Et(3)P-Tu species, characterized by delta 31P of 1.0 ppm, assigned after an independent reaction between Et(3)P and Tu. In an experiment using a 0.05 M solution of auranofin, the Et(3)PO resonance appeared in auranofin spectrum after 4 days of addition of 1.0 equivalent of Tu, showing that the reaction is slow. A resonance for free Et(3)P is also detected in 31P NMR on the addition of CN(-). It is also observed that Tu reacts with Et(3)PAuCl to form [Et(3)P-Au-Tu](+) via displacement of Cl(-), consistent with an upfield shift of 6.2 ppm in >C [double bond] S resonance of Tu in (13)C NMR. In (15)N NMR, a smaller downfield, instead of an upfield shift, in NH(2) resonance of Tu on its addition to auranofin and Et(3)PAuCl indicates that it is not binding to gold(I) through nitrogen.     

Natural abundance 15N NMR assignments delineate structural differences between intact and reactive-site hydrolyzed Cucurbita maxima trypsin inhibitor III.

15N NMR assignments were made to the backbone amide nitrogen atoms at natural isotopic abundance of intact and reactive-site (Arg5-Ile6) hydrolyzed Cucurbita maxima trypsin inhibitor III (CMTI-III and CMTI-III*, respectively) by means of 2D proton-detected heteronuclear single bond chemical shift correlation (HSBC) spectroscopy, utilizing the previously made sequence-specific 1H NMR assignments (Krishnamoorthi et al. (1992) Biochemistry 31, 898-904). Comparison of the 15N chemical shifts of the two forms of the inhibitor molecule revealed significant changes not only for residues located near the reactive-site region, but also for those distantly located. Residues Cys3, Arg5, Leu7, Met8, Cys10, Cys16, Glu19, His25, Tyr27, Cys28 and Gly29 showed significant chemical shift changes ranging from 0.3 to 6.1 ppm, thus indicating structural perturbations that were transmitted throughout the molecule. These findings confirm the earlier conclusions based on 1H NMR investigations.     

High-resolution phosphorus nuclear magnetic resonance spectroscopy of transfer ribonucleic acids: multiple conformations in the anticodon loop

The temperature dependence of the 31P NMR spectra of yeast phenylalanine tRNA, E. coli tyrosine, glutamate (2), and formylmethionine tRNA is presented. The major difference between the 31P NMR spectra of the different acceptor tRNAs is in the main cluster region between -0.5 and -1.3 ppm. This confirms an earlier assignment of the main cluster region to the undistorted phosphate diesters in the hairpin loops and helical stems. In addition the 31P NMR spectra for all tRNAs reveal approximately 16 nonhelical diester signals spread over approximately 7 ppm besides the downfield terminal 3'-phosphate monoester. In the presence of 10 mM Mg2+ most scattered and main cluster signals do not shift between 22 and 66 degrees C, thus supporting our earlier hypothesis that 31P chemical shifts are sensitive to phosphate ester torsional and bond angles. At greater than 70 degrees C, all of the signals merge into a single random-coil conformation signal. A number of the scattered peaks are shifted (0.2-1.7 ppm) and broadened between 22 and 66 degrees C in the presence of Mg2+ and spermine as a result of a conformational transition in the anticodon loop. The 31P NMR spectrum of the dimer formed between yeast tRNAPhe and E. coli tRNA 2Glu is reported. This dimer simulates codon-anticodon interaction since the anticodon triplets of the two tRNAs are complementary. Evidence is presented that the anticodon-anticodon interaction alters the anticodon conformation and partially disrupts the tertiary structure of the tRNA.     

Lumazine proteins from photobacteria: localization of the single ligand binding site to the N-terminal domain

Lumazine protein is believed to serve as an optical transponder in bioluminescence emission by certain marine bacteria. Sequence arguments suggest that the protein comprises two similarly folded riboflavin synthase-type domains, but earlier work also suggested that only one domain binds 6,7-dimethyl-8-ribityllumazine (DMRL). We show that the replacement of serine-48 or threonine-50 in the N-terminal domain of lumazine protein of Photobacterium leiognathi modulates the absorbance and fluorescence properties of bound DMRL or riboflavin. Moreover, the replacement of these amino acids is accompanied by reduced ligand affinity. Replacement of serine-48 by tryptophan shifts the (13)C NMR signal of the 6-methyl group in bound DMRL upfield by 2.9 ppm as compared to the wild-type protein complex. Replacement of threonine-50 causes a downfield shift of approximately 20 ppm for the (15)N NMR signal of N-5, as well as an upfield shift of 3 ppm for the (13)C NMR signal of C-7 in bound DMRL, respectively. The replacement of the topologically equivalent serine-144 and proline-146 in the C-terminal domain had no significant impact on optical properties, chemical shifts and apparent binding constants of bound DMRL. These data show that the N-terminal domain is the unique site for ligand binding in lumazine protein.     

Binding of oxytocin and 8-arginine-vasopressin to neurophysin studied by 15N NMR using magnetization transfer and indirect detection via protons

NMR was used to monitor the binding to neurophysin of oxytocin and 8-arginine-vasopressin, 15N labeling being used to identify specific backbone 15N and 1H signals. The most significant effects of binding were large downfield shifts in the amino nitrogen resonance of Phe-3 of vasopressin and in its associated proton, providing evidence that the peptide bond between residues 2 and 3 of the hormones is hydrogen-bonded to the protein within hormone-neurophysin complexes. Suggestive evidence of hydrogen bonding of the amino nitrogen of Tyr-2 was also obtained in the form of decreased proton exchange rates on binding; however, the chemical shift changes of this nitrogen and its associated proton indicated that such hydrogen bonding, if present, is probably weak. Shifts in the amino nitrogen of Asn-5 and in the -NH protons of both Asn-5 and Cys-6 demonstrated that these residues are significantly perturbed by binding, suggesting conformational changes of the ring on binding and/or the presence of binding sites on the hormone outside the 1-3 region. No support was obtained for the thesis that there is a significant second binding site for vasopressin on each neurophysin chain. The behavior of both oxytocin and vasopressin on binding was consistent with formation of 1:1 complexes in slow exchange with the free state under most pH conditions. At low pH there was evidence of an increased exchange rate. Additionally, broadening of 15N resonances in the bound state at low pH occurred without a corresponding change in the resonances of equilibrating free hormone.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMR chemical shift mapping of the binding site of a protein proteinase inhibitor: changes in the (1)H, (13)C and (15)N NMR chemical shifts of turkey ovomucoid third domain upon binding to bovine chymotrypsin A(alpha)

The substrate-like inhibition of serine proteinases by avian ovomucoid domains has provided an excellent model for protein inhibitor-proteinase interactions of the standard type. 1H,15N and 13C NMR studies have been undertaken on complexes formed between turkey ovomucoid third domain (OMTKY3)2 and chymotrypsin A(alpha) (Ctr) in order to characterize structural changes occurring in the Ctr binding site of OMTKY3. 15N and 13C were incorporated uniformly into OMTKY3, allowing backbone resonances to be assigned for OMTKY3 in both its free and complex states. Chemical shift perturbation mapping indicates that the two regions, K13-P22 and N33-A40, are the primary sites in OMTKY3 involved in Ctr binding, in full agreement with the 12 consensus proteinase-contact residues of OMTKY3 defined previously on the basis of X-ray crystallographic and mutational analysis. Smaller chemical shift perturbations in selected other regions may result from minor structural changes on binding. Through-bond 15N-13C correlations between P1-13C' and P1'-15N in two-dimensional H(N)CO and HN(CO) NMR spectra of selectively labeled OMTKY3 complexed with Ctr indicate that the scissile peptide bond between L18 and E19 of the inhibitor is intact in the complex. The chemical shifts of the reactive site peptide bond indicate that it is predominantly trigonal, although the data are not inconsistent with a slight perturbation of the hybridization of the peptide bond toward the first tetrahedral state along the reaction coordinate.     

On the coordination of inhibitors to the metal ion of carboxypeptidase A. A 113Cd and 31P NMR study

113Cd and 31P NMR have been used to investigate the interactions of inhibitors with the metal ion of bovine carboxypeptidase A, using 113Cd as a replacement for the native zinc atom. In the absence of inhibitor and over the pH range 6-9, no 113Cd resonance is visible at room temperature. Upon lowering the temperature to 270 K, however, a broad resonance can be seen at 120 ppm. These results are discussed in terms of possible sources for this resonance modulation. Binding of low molecular weight inhibitors containing potential metal-coordinating moieties results in the appearance of a sharp 113Cd resonance. These inhibitors all bind to the metal ion, a fact which is reflected in the chemical shift of the cadmium resonance and, for L-phenylalanine phosphoramidate phenyl ester, by two-bond 113Cd-31P spin-spin coupling of 30 Hz in the 31P resonance of the bound inhibitor. For inhibitors that coordinate to the metal ion via oxygen, the 113Cd chemical shift is in the range 127-137 ppm, whereas for sulfur coordination there is a downfield shift of approximately 210 ppm. The complexes of 113Cd-substituted carboxypeptidase A with the D and L isomers of thiolactic acid are distinguished by a difference of 11 ppm in the chemical shift of their cadmium resonances. The enzyme complex formed with the macromolecular inhibitor from potatoes, which fills the S1 and S2 subsites, shows one or possibly two closely spaced broad 113Cd resonances. Both the chemical shift and the line width of the 113Cd resonances of the [113Cd]carboxypeptidase-inhibitor complexes give valuable structural and dynamic information about the enzyme active site.