Structure of the ribotrinucleoside diphosphate codon UpUpC bound to tRNAPhe from yeast. A time-dependent transferred nuclear Overhauser enhancement study

The structure of the ribotrinucleoside diphosphate UpUpC, the codon for phenylalanine, bound to yeast tRNAPhe in solution is elucidated using time-dependent proton-proton transferred nuclear Overhauser enhancement measurements to determine distances between bound ligand protons. The glycosidic bond and ribose conformations are low anti and 3'-endo, respectively, typical of an A-RNA type structure. The main chain torsion angles are all within the range of those expected for A-RNA but small differences from those in conventional A-RNA 11 result in a special structure with a larger rotation per residue (40 to 45 degrees compared to 32.7 degrees in R-RNA 11) and almost perfect stacking of the bases. These two structural features, which are similar to those found in the anticodon triplet of the monoclinic crystal form of tRNAPhe, can account for the known greater stability of the codon-anticodon complex relative to an equivalent double helical RNA trimer with a conventional A-RNA structure.     

Accuracy of bound peptide structures determined by exchange transferred nuclear Overhauser data: A simulation study

The exchange-transferred NOE method to determine the three-dimensional structure of peptides bound to proteins, or other macromolecular systems, is becoming increasingly important in drug design efforts and for large or multicomponent assemblies, such as membrane receptors, where structural analysis of the full system is intractable. The exchange-transferred nuclear Overhauser effect spectroscopy (etNOESY) method allows the determination of the bound-state conformation of the peptide from the intra-molecular NOE interactions between ligand protons. Because only ligand–ligand NOEs are generally observable, the etNOESY method is restricted to fewer NOEs per residue than direct protein structure determination. In addition, the averaging of relaxation rates between free and bound states affects the measured cross-peak intensities, and possibly the accuracy of distance estimates. Accordingly, the study reported here was conducted to examine the conditions required to define a reliable structure. The program CORONA was used to simulate etNOE data using a rate-matrix including magnetic relaxation and exchange rates for two peptide–protein complexes derived from the reference complex of cAMP-dependent protein kinase ligated with a 24-residue inhibitor peptide. The results indicate that reasonably accurate peptide structures can be determined with relatively few NOE interactions when the interactions occur between non-neighboring residues. The reliability of the structural result is suggested from the pattern of NOE interactions. A structure with an accuracy of approximately 1.3 Å rms difference for the main-chain atoms can be obtained when etNOE interactions between non-neighboring residues occur over the length of the peptide. The global precision is higher (approximately 0.9 Å rms difference) but is not correlated to global accuracy. A local definition of precision along the backbone appears to be a good indicator of the local accuracy.     

Analysis of an enzyme-substrate complex by X-ray crystallography and transferred nuclear Overhauser enhancement measurements: porcine pancreatic elastase and a hexapeptide

The hexapeptide substrate Thr-Pro-nVal-NMeLeu-Tyr-Thr reacts with porcine pancreatic elastase sufficiently slowly that accelerated crystallographic data collection procedures and two-dimensional transferred nuclear Overhauser enhancement measurements could be used to study the geometry of binding. Both studies report a time-averaged population of the Michaelis complex state, prior to proteolysis. This result provides an important data point along the reaction coordinate pathway for serine proteases. Crystallographic data to 1.80-A resolution were used in the structure analysis with refinement to an R-factor of 0.19.     

Internal mobility in a double-stranded B DNA hexamer and undecamer. A time-dependent proton-proton nuclear Overhauser enhancement study

The internal mobility of the deoxyribose H2'-H2" and base C(H5)-C(H6) and T(CH3)-T(H6) vectors has been investigated by means of time-dependent nuclear Overhauser enhancement (NOE) measurements in a B DNA hexamer and undecamer. Cross-relaxation rates between these proton pairs are determined from the initial slopes of the time development of the NOEs, and, as the interproton distances between these proton pairs are fixed, apparent correlation times for the 3 interproton vectors are calculated from the cross-relaxation rate data. It is shown that there is little residue to residue variation in the cross-relaxation rates of the interproton vectors within each oligonucleotide, that the mean apparent correlation times of the C(H5)-C(H6) and T(CH3)-T(H6) vectors are approximately equal and significantly greater than that of the H2'-H2" vectors, and that the data for the H2'-H2" vectors of both oligonucleotides and the C(H5)-C(H6) and T(CH3)-T(H6) vectors of the undecamer cannot be accounted for by isotropic tumbling alone. The data are analysed in terms of a two motion model with isotropic tumbling and a single internal motion. The relaxation time of the internal motion at 23 degrees C is less than or equal to 1 ns for the H2'-H2" vectors of both oligonucleotides and less than or equal to 3 ns for the C(H5)-C(H6) and T(CH3)-T(H6) vectors of the undecamer. In the case of the H2'-H2" vectors, however, the amplitude of the internal motion is found to be too large to be compatible with the known stereochemistry of DNA. This finding can only be explained by invoking additional degrees of internal freedom with a larger number of internal motions of small amplitude of the type deduced from the analysis of crystallographic thermal factors [(1984) J. Mol. Biol. 173, 361-388].     

Structure of antibody-bound peptides and retro-inverso analogues. A transferred nuclear Overhauser effect spectroscopy and molecular dynamics approach

The three-dimensional structures of the two L-peptides, H-CGGIRGERA-OH, called L(A), and H-CGGIRGERG-OH, called L(G), corresponding or close to the IRGERA sequence present in the C-terminal region (residues 130-135) of histone H3, and their retro-inverso analogues HO-mAreGriGGC-NH2, called RI(mA), and HO-mGreGriGGC-NH2, called RI(mG), have been studied by two-dimensional 1H NMR and molecular dynamics calculations in association with a monoclonal antibody generated against L(A). At 25 degrees C, the affinity constants of the monoclonal antibody with respect to RI(mA) and RI(mG) were 75- and 270-fold higher than those measured with the homologous L(A) and L(G) peptides, respectively. Due to the spontaneous epimerization of the mA malonic residue, RI(mA) gave rise to two sets of resonances. With regard to the NH amide region, one set was similar to that for RI(mG) while the second was similar to those for the parent L-peptides L(A) and L(G). The antibody-bound conformations of the two couples of L- and retro-inverso peptides have been analyzed using molecular modeling calculations based on the transferred NOE interproton distances. Folded structures appeared in both cases with a type II' beta-turn in the parent GGIR sequence and a type I' beta-turn in the retro-inverso reGr sequence.     

Conformation of the DNA undecamer 5'd(A-A-G-T-G-T-G-A-T-A-T) bound to the single-stranded DNA binding protein of Escherichia coli. A time-dependent transferred nuclear Overhauser enhancement study

A time-dependent transferred nuclear Overhauser enhancement study of the conformation of the single-stranded DNA 11mer 5'd(A-A-G-T-G-T-G-A-T-A-T) bound to the single-stranded DNA binding protein of Escherichia coli (SSB) is presented. It is shown that the conformation of the bound 11mer is that of a right-handed B-type helix similar to that of the free 11mer. The observation of internucleotide transferred nuclear Overhauser enhancements for every base step excludes the possibility of intercalation by aromatic protein residues. In addition, it is shown that the effective correlation time of the bases (80 ns) corresponds to that of a complex of molecular weight approximately 170,000, containing two SSB tetramers. The sugars, on the other hand, exhibit a shorter effective correlation time (40 ns), indicating the presence of internal motion. This suggests that the bases are anchored to the protein surface, possibly by hydrophobic interactions, whereas the sugar-phosphate groups are directed outwards towards the solvent.     

Conformation of NAD+ bound to yeast and horse liver alcohol dehydrogenase in solution. The use of the proton-proton transferred nuclear Overhauser enhancement

Changes in reduced viscosity of nuclear lysates from rat liver cells have been studied, in conditions of very low shear stress by the use of an oscillating viscometer, as a function of incubation time in alkaline (pH 12.5) and neutral (pH 8.0) solutions. In non-denaturing conditions, nuclear DNA showed a stepwise, time-dependent increase of reduced viscosity, which suggests that it behaves as a single hydrodynamic unit that progressively changes its radius and viscoelastic properties because of a very slow unfolding, through discrete successive transitions, from a highly superpacked structure toward a linear relaxed B-form fiber. Experimental conditions shown to reduce chromatin-DNA superpacking without changing DNA length (e.g. G₁ cycling versus G₀ non-cycling liver cells, or young versus old rat liver cells) dramatically increased the initial value of reduced viscosity and its time-dependent increment. Conversely, in denaturing conditions, reduced viscosity increased in the initial phase (probably because DNA unfolding prevails on DNA unwinding), then exhibited a plateau level (when unfolding balances unwinding), and subsequently decreased progressively to the value of sheared DNA (when unwinding becomes more rapid due to the progressive breakage of phosphodiester bridges in alkali). Experimental conditions known to induce DNA single- or double-strand breaks (i.e. the use of liver cells from rats treated with dimethylnitrosamine or 2-acetylaminofluorene, or of liver cells exposed to X-rays) caused in both neutral and alkaline solution an increment in the initial reduced viscosity and in the slope of its time-dependent increase, which may be related to a reduction of chromatin-DNA superpacking. Moreover, it became evident in denaturing conditions that a decrease of the maximum viscosity and of the time taken to reach it both related to a reduced DNA length. These viscoelastic properties are constantly correlated with independent DNA structural measurements on the same nuclear lysates, to discriminate the effect due to mere aggregation and disaggregation.     

Wobble base-pairing in codon-anticodon interactions: a theoretical modelling study

The Crick wobble hypothesis attributes the phenomenon of codon degeneracy to a certain impreciseness of pairing between the third base of the codon and the first base of the anticodon. This theoretical study investigates the pairing properties of some wobble bases, including both, observed and unobserved pairs. Some wobble base-pairs are predicted to follow the Watson-Crick pairs in configuration and pairing facility, while others deviate from this norm. The observed U:V pair is unique in that a pairing configuration may be suggested for it wherein the hydrogen-bonding involves the exocyclic 5-carboxymethoxy group of V. By comparing the theoretical data on the configurations of these pairs with the evidence for their existence/non-existence in nature, some guidelines emerge for differentiating between observed and unobserved base pairs on the basis of the pairing configuration.     

Adenosine conformations of nucleotides bound to methionyl tRNA synthetase by transferred nuclear Overhauser effect spectroscopy.

The conformations of MgATP and AMP bound to a monomeric tryptic fragment of methionyl tRNA synthetase have been investigated by two-dimensional proton transferred nuclear Overhauser effect spectroscopy (TRNOESY). The sample protocol was chosen to minimize contributions from adventitious binding of the nucleotides to the observed NOE. The experiments were performed at 500 MHz on three different complexes, E.MgATP, E.MgATP.L-methioninol, and E.AMP.L-methioninol. A starter set of distances obtained by fitting NOE build-up curves (not involving H5' and H5") were used to determine a CHARMm energy-minimized structure. The positioning of the H5' and H5" protons was determined on the basis of a conformational search of the torsion angle to obtain the best fit with the observed NOEs for their superposed resonance. Using this structure, a relaxation matrix was set up to calculate theoretical build-up curves for all of the NOEs and compare them with the observed curves. The final structures deduced for the adenosine moieties in the three complexes are very similar, and are described by a glycosidic torsion angle (chi) of 56 degrees +/- 5 degrees and a phase angle of pseudorotation (P) in the range of 47 degrees to 52 degrees, describing a 3(4)T-4E sugar pucker. The glycosidic torsion angle, chi, deduced here for this adenylyl transfer enzyme and those determined previously for three phosphoryl transfer enzymes (creatine kinase, arginine kinase, and pyruvate kinase), and one pyrophosphoryl enzyme (PRibPP synthetase), are all in the range 52 degrees +/- 8 degrees. The narrow range of values suggests a possible common motif for the recognition and binding of the adenosine moiety at the active sites of ATP-utilizing enzymes, irrespective of the point of cleavage on the phosphate chain.     

Conformation of beta-methylmelibiose bound to the ricin B-chain as determined from transferred nuclear Overhauser effects.

Transferred nuclear Overhauser effect (TRNOE) experiments have revealed a change in the torsion angles about the alpha-1-6 glycosidic bond of methyl beta-melibioside upon binding of the melibioside to the ricin B-chain (Rb). A full relaxation rate matrix simulation of experimental buildup curves aided in quantitative interpretation of 1D selective inversion recovery TRNOE experiments. The data are consistent with a model in which both major (omega approximately 170 degrees) and minor (omega approximately -60 degrees) conformers for methyl beta-melibioside are significantly populated in solution while the Rb/methyl beta-melibioside complex has little of the minor conformer populated. The results indicate that the ricin B-chain excludes binding of certain ligand conformations on the basis of unfavorable interactions between the protein surface and remote portions of the disaccharide system.     

Chloroplast genes transferred to the nuclear plant genome have adjusted to nuclear base composition and codon usage.

During plant evolution, some plastid genes have been moved to the nuclear genome. These transferred genes are now correctly expressed in the nucleus, their products being transported into the chloroplast. We compared the base compositions, the distributions of some dinucleotides and codon usages of transferred, nuclear and chloroplast genes in two dicots and two monocots plant species. Our results indicate that transferred genes have adjusted to nuclear base composition and codon usage, being now more similar to the nuclear genes than to the chloroplast ones in every species analyzed.     

Scope and limitations of rotating-frame nuclear Overhauser enhancement spectroscopy applied to oligosaccharides

Two-dimensional rotating-frame nuclear Overhauser enhancement spectroscopy has been applied to milk oligosaccharides containing the blood-group determinants Lea, Led, or Leb. The results indicate a large number of dipolar interactions that are consistent with the preferred conformations described for these blood-group determinants. Despite careful experimental conditions, coherent transfer processes are not suppressed completely, allowing only a qualitative evaluation of the nuclear Overhauser data. The 1H- and 13C-n.m.r. spectra are assigned by comparison with literature data and by application of two-dimensional 13C-1H shift-correlation spectroscopy.     

The use of transferred nuclear Overhauser effects in the study of the conformations of small molecules bound to proteins

A novel method is proposed for the study of the conformation in solution of small molecules bound to proteins. In transfer of saturation experiments, irradiation at the frequency of a proton in the bound ligand can result in an intensity change in the signal from a different proton in the free excess ligand via a nuclear Overhauser effect between the two protons in the bound ligand. Approximatel calculations show that the observation of such effects depends upon the close spatial proximity (within about 4.0 Å) of the two protons involved and thus gives useful conformational information. Two examples of this method are given, for the binding of trimethoprim and NADP⁺, respectively, to Lactobacillus casei dihydrofolate reductase.     

Nuclear Overhauser effect study and assignment of D stem and reverse-Hoogsteen base pair proton resonances in yeast tRNAAsp

Nuclear Overhauser effects (NOEs) in yeast tRNAAsp were found for all four GU and G psi base pairs. NOEs of both reverse-Hoogsteen pairs were identified by comparison with a purine C8 deuterated sample. Several NOEs involving these resonances were also found which are clearly between single protons on adjacent base pairs. These interbase NOEs, combined with the assumption of reasonable similarity between the structure of yeast tRNAAsp and that of yeast tRNAPhe, lead to unambiguous assignment of many resonances including all the ring NH and C2 protons in the D stem. The stability of the stem at 28 degrees C, as recently deduced by Moras et al (Nature 288 669-674), from x-ray diffraction is confirmed. Assignments of the ring NH resonances of T54-A58 and of a G psi pair are made for the first time.     

Nuclear magnetic resonance studies of flexible opiate conformations at monoclonal antibody binding sites. Quantitative interproton distances obtained from comparing theoretical and experimental transferred nuclear Overhauser enhancement: correlation with antibody sequence

A previous publication described the use of qualitative intramolecular 1H-transferred nuclear Overhauser effect measurements to determine the conformations of flexible ligands at monoclonal anti-opiate antibody binding sites. This paper concentrates on the quantitative interpretation of experiments of this type using the ligand nalorphine (N-allyl morphine) and a single anti-opiate monoclonal antibody. I compare the experimental unidimensional driven nuclear Overhauser effect buildup curves to theoretical curves derived with a knowledge of the fixed interproton distances in the ligand. The discussion covers the potential accuracies of derived distances and concentrates on two problem areas associated with determining structures from this type of experiment. The most serious one is the case where, because of particular multiproton spatial distributions, spin diffusion is so rapid that it cannot be determined experimentally and where numerical fits of theoretical calculations are misleading. The results show that, while intraligand spin diffusion complicates the interpretation for some proton pairs, with many others accuracies within about 0.3 A for interproton distances from 2 to 4 A are attainable. The results confirm the earlier report that the conformation of nalorphine in this antibody binding site differs from the major one present in solution or in the crystal. An important aspect of the work is that theoretical prediction of nuclear Overhauser effect time-dependence is an important practical tool for recognizing cases where interpretation of experiments will be difficult. Initial data on protein-to-ligand transferred nuclear Overhauser effect are presented, which show that at least one aromatic amino acid residue is closely involved in the binding of the ligand. The companion paper presents the primary sequences of the variable regions of the antibodies being used in our studies. In this paper, these and associated immunochemical studies are correlated with the nuclear magnetic resonance results. The combination of data presented in the two papers provides a basis for future work on protein-ligand interproton distances in the range 1 to 5 A using both transferred nuclear Overhauser effect (for rapidly exchanging ligands) and isotope-edited, indirectly detected nuclear Overhauser effect (for tightly bound ligands).     

A conformational study of some adenosines by use of nuclear Overhauser effect

Conformations of 8-bromo-2′-[unk]-triisopropylbenzenesulfonyladenosine ([unk]) and its 3′-[unk]-isomer ([unk]) in solution have been determined by the use of intramolecular nuclear Overhauser effects in ¹H NMR spectroscopy. Compound [unk] has been proved to have a conformation in which the adenosine and benzene rings are intramolecularly stacked and compound [unk] an elongated non-stacked conformation in dimethylsulphoxide. The 5′-[unk]-acetyl derivative of [unk] has also been found to adopt the intramolecularly stacked conformation in dimethylsulphoxide, but a non-stacked one in chloroform. Coupling constants observed are discussed in connection with the conformation of the ribose moiety. The ¹³C NMR spectra have also been examined, but no effect which could be ascribed to the stacking phenomena was observed in the carbon chemical shifts.     

Conformations of bound nucleoside triphosphate effectors in aspartate transcarbamylase. Evidence for the London-Schmidt model by transferred nuclear Overhauser effects

Transferred nuclear Overhauser effects were used to determine the conformations of ATP, CTP, and ITP bound to the regulatory site of aspartate transcarbamylase. The results are in accord with the predictions of the London-Schmidt model [London, R. E., & Schmidt, P. G. (1972) Biochemistry 11, 3136] and show that ATP and CTP bind in the anti conformation while ITP binds in the syn conformation.     

Stereochemistry of binding of the tetrapeptide acetyl-Pro-Ala-Pro-Tyr-NH2 to porcine pancreatic elastase. Combined use of two-dimensional transferred nuclear Overhauser enhancement measurements, restrained molecular dynamics, X-ray crystallography and molecular modelling

A nuclear magnetic resonance study of the conformation of the tetrapeptide acetyl-Pro-Ala-Pro-Tyr-NH2 bound to porcine pancreatic elastase is presented. From two-dimensional transferred nuclear Overhauser enhancement measurements, a set of 23 approximate distance restraints between pairs of bound ligand protons, indicative of an extended type structure, is derived. The structure of the bound tetrapeptide is then refined from two different starting structures (an extended beta-strand and a polyproline helix) by restrained molecular dynamics, in which the interproton distances are incorporated into the total energy of the system in the form of effective potentials. Convergence to essentially the same average restrained dynamics structures is achieved. The refined structures are then modelled into the active site of elastase by interactive molecular graphics. The determination of the anchor point of the bound tetrapeptide on the enzyme was aided by a simultaneous crystallographic study which, despite the fact that only electron density for a Pro-X dipeptide fragment was visible, enabled both the approximate position and orientation of binding to be determined. It is found that the tetrapeptide is bound in the S' binding site in the reverse orientation found in other serine protease-inhibitor complexes and is stabilized both by hydrogen-bonding and by van der Waals' interactions.