Two-dimensional 1H nuclear magnetic resonance studies on the gene V-encoded single-stranded DNA-binding protein of the filamentous bacteriophage IKe. I. Structure elucidation of the DNA-binding wing

Two-dimensional nuclear magnetic resonance techniques were used to obtain residue- and sequence-specific assignments in the 1H spectrum of the single-stranded DNA-binding protein encoded by gene V of the filamentous phage IKe (IKe GVP). The residue-specific assignments are based on the analysis of J-correlated spectra, i.e. correlated spectroscopy and homonuclear-Hartmann-Hahn total correlated spectroscopy. Complete assignments of side-chain spin systems, e.g. long side-chains, were, to a major part, derived from two-dimensional spectra obtained by means of the latter technique. Sequence-specific residue assignments were obtained for the two neighbouring residues V41 and Y42, and the amino acid sequence segment encompassing residues S17 through I29. The structure of this segment, a beta-loop, was deduced from the interresidue nuclear Overhauser effect pattern. Residues S17 through V19 and P26 through I29 form an anti-parallel beta-ladder segment, whereas residues Q21 to K25 constitute the loop region. The beta-loop is expected to project into the solution and is intimately involved in binding to single-stranded DNA; it is therefore designated the "DNA-binding wing". By analogy with the structure of the DNA-binding wing deduced from IKe GVP, a similar structure is proposed for the corresponding domain of the gene V protein encoded by the filamentous phage Ff for which, from X-ray diffraction studies, a three-dimensional structure has been deduced. Essential differences appear to exist between the DNA-binding domain in the X-ray structure and that proposed in this paper. Possible reasons for these differences are discussed.     

1H, 15N, 13C resonance assignments of the reduced and active form of human Protein Tyrosine Phosphatase, PRL-1

Phosphatase of regenerating liver-1 (PRL-1) is a novel target for potentially treating cancer metastases. Although its specific biochemical role in these processes has yet to be delineated, considerable evidence suggests the phosphatase activity of PRL-1 is required for promoting cancer and metastasis. PRL-1 belongs to the protein tyrosine phosphatase (PTPase) family and functions using the CX₅R consensus active site motif. Like other PTPases, PRL-1 is inhibited by oxidation at its active site Cys, however, disulfide bond formation occurs unusually readily in wild-type PRL-1. Chemical shift assignments are available for oxidized wild type, but numerous, substantial changes are observed in the spectra upon reduction. Because the reduced form is active, we sought to identify a stable mutant that would resist oxidation and be useful for facilitating drug screening and development using NMR-based assays. We present here NMR assignments for a full-length, reduced and active form of PRL-1, PRL-1-C170S-C171S, that is well suited for this purpose.     

Assignment of resonances in the downfield proton spectrum of Escherichia coli 5S RNA and its nucleoprotein complexes using components of a ribonuclease-resistant fragment

The downfield (9-15 ppm) proton NMR spectra of oligonucleotides derived from the ribonuclease A resistant fragment of Escherichia coli 5S RNA have been examined in aqueous solution at 500 MHz. Comparison of these spectra with those of the 5S RNA fragment and intact 5S RNA using both chemical shift and nuclear Overhauser enhancement effect criteria indicates that several aspects of 5S RNA secondary structure are also present in the structures assumed in solution by these much smaller molecules. Analysis of these spectra permits the assignment of some imino proton resonances which could not be assigned with certainty on the basis of NMR data previously obtained on intact 5S RNA or its nucleoprotein complexes. Several previous resonance assignments are confirmed. Studies on oligonucleotide components of fragment and a reconstituted fragment show that at least two conformations of the procaryotic loop exist.     

NMR assignments of the four histidines of staphylococcal nuclease in native and denatured states

NMR signals from all four histidine ring C epsilon protons and three of the four histidine C delta protons in the protein staphylococcal nuclease have been assigned by comparing spectra of the wild-type (Foggi strain) protein to spectra of three variants that each lack a different histidine residue. All proteins studied were cloned and overproduced in Escherichia coli. The NMR spectra of the three mutant proteins (H8R, H46Y, and H124L) used to make these assignments were similar to one another and to those of the wild type, except for signals from the mutated residues. The pKa values of those histidines conserved between the wild type and the mutants remained essentially unchanged. Multiple histidine C epsilon proton resonances due to non-native forms of nuclease were observed in both thermally induced and acid-induced unfolding. Residue-specific assignments of H epsilon protons in the thermally denatured forms of the mutant H46Y were obtained from connectivities to the native state by saturation transfer.     

1H, 13C, and 31P nuclear magnetic resonance spectral assignments for tobramycin, 2'-(adenosine-5'-phosphoryl)-tobramycin and 2'-(adenosine-5'-thiophosphoryl)-tobramycin

Two enzymatically modified derivatives of tobramycin have been prepared by gentamicin nucleotidyl transferase-catalyzed adenylylation of tobramycin, using ATP and (Sp)-ATP alpha S as adenylylation substrates. (EC 2.7.7.46). The 1H, 13C, and 31P NMR spectra have been assigned for tobramycin, 2'-(adenosine-5'-phosphoryl)-tobramycin (TbAMP) and 2'-(adenosine-5'-thiophosphoryl)-tobramycin (TbAMPS). Several one- and two-dimensional NMR techniques have been utilized, notably, 1H-1H homonuclear correlation spectroscopy at 470 or 500 MHz and 13C-1H heteronuclear correlation spectroscopy at 50.3 MHz. The 1H assignments for tobramycin are similar to those previously reported for rings I and III of kanamycin A. The 13C assignments for tobramycin were similar to those previously reported, except for reversal of the assignments for anomeric carbons in the glycosyl rings. The 1H and 13C assignments for tobramycin were used to guide the assignments of the spectra for TbAMP and TbAMPS. Nearly complete assignments were obtained for these two derivatives of tobramycin. From the measured proton coupling constants, only small conformational changes were observed upon modification of tobramycin by adenylylation. From the proton and carbon spectra of the adenylylated derivatives the 2' position is shown to be the site of adenylation. Large downfield shifts of the 2'proton and carbon resonances are easily observed and are more pronounced for TbAMPS than for TbAMP.     

1H, 13C and 15N resonance assignments of S114A mutant of UVI31+ from Chlamydomonas reinhardtii

Almost complete sequence specific ¹H, ¹³C and ¹⁵N resonance assignments of S114A mutant of UVI31+ from Chlamydomonas reinhardtii are reported. The cDNA of S114A mutant of UVI31+ was cloned from a eukaryotic green algae (C. reinhardtii) and overexpressed in E.coli from where the protein was purified to homogeneity. The point mutation S114A in UVI31+ reduces its DNA endonuclease activity substantially as compared with its wild type. As a prelude to the structural characterization of S114A mutant of UVI31+, we report here complete sequence-specific ¹H, ¹³C and ¹⁵N NMR assignments.     

1H-NMR assignments and local environments of aromatic residues in bovine, human and guinea pig variants of alpha-lactalbumin.

1H-NMR assignments have been defined for the aromatic-ring protons of the bovine, guinea pig and human variants of alpha-lactalbumin. Spin-system networks were identified by means of double-quantum-filtered two-dimensional J-correlated spectroscopy and two-dimensional relayed coherence spectroscopy data. Analysis of two-dimensional nuclear-Overhauser-enhancement spectroscopy data of the proteins indicated that in each case two clusters of aromatic residues exist. The two clusters are also evident in the crystal structure of the human protein, and this evidence, in conjunction with sequence differences between the three proteins, permitted sequence-specific assignments to be made for the majority of aromatic residues. Remaining ambiguities in the assignments could be resolved by analysis of photochemically induced dynamic nuclear polarization (PCIDNP) effects. Comparison of the PCIDNP spectra of the three proteins indicated the presence of only minor differences in the surface exposure of conserved aromatic residues. Taken together, these results indicate that the environments of the conserved aromatic residues in bovine, guinea pig and human alpha-lactalbumin in solution are very similar to each other, and that the solution and the crystal forms of at least the human protein are similar.     

NMR signal assignments of amide protons in the alpha-helical domains of staphylococcal nuclease

We report complete assignments of the amide proton signals in the three long dNN connectivity sequences observed in the NOESY spectrum of deuteriated staphylococcal nuclease (Nase) complexed with thymidine 3',5'-bisphosphate (pdTp) and Ca2+, Mr 18K. The assignments are made by comparing NOESY spectra with 1H-15N and 1H-13C heteronuclear multiple-quantum shift correlation (HMQC) spectra of Nase samples containing 15N- and 13C-labeled amino acids. The assignments show that the residues which are linked by the dNN connectivity sequences are located in three alpha-helical domains of Nase. Our results indicate that by combining NOESY and HMQC spectra of appropriately labeled samples it should be possible to delineate and study alpha-helical domains in soluble proteins having molecular weights that are greater than 18K.     

Far-Infrared Study of the Vibrational Modes of 5′-GMP Gels and Crystals of Na+ and K+

Abstract

Five Far-Infrared (50–600 cm^?1) spectra are presented: the sodium and potassium salts of 5′ Guanosine Monophosphate (GMP), each salt in both the gel and crystal conformations, and poly(rG). Measurements were performed at a sample temperature of 10 Kunder vacuum with a liquid He-cooled bolometer. The spectra were fit with Lorentzians and assignments are suggested. There are noteworthy differences in oscillator strengths and frequencies of the bands between all spectra. We report the tentative observation of a 100 cm^?1 mode which is in the neighborhood of a mode observed by Raman spectroscopy in solution (1) and dried gels (2).     

Preparation of trimers and tetramers of mixed sequence oligodeoxynucleoside methylphosphonates and assignment of configurations at the chiral phosphorus.

Synthesis of stereoregular DNA methylphosphonates has been accomplished for homo-oligomers, but remains a formidable problem for oligomers of a defined antisense target sequence. In this work, four trimer and tetramer deoxynucleoside methylphosphonates of mixed sequence (dACA, dCCAA, dAGGG, and dGCAT) were prepared by block coupling of diastereomerically pure dimers with either monomers or other diastereomerically pure dimers. These oligomers were separated chromatographically into individual diastereomers, and the configurations of the chiral methylphosphonate linkages were assigned. Three types of methods were used to assign configuration of a new methylphosphonate linkage: preparation of the same diastereomer through multiple synthetic pathways, base hydrolysis, and acid hydrolysis. Hydrolysis of the diastereomerically pure oligomers into component dimers and monomers was followed by chromatographic comparison with control dimers of known configuration. In all cases studied, oligomers with R configurations displayed faster elution from silica gel than did oligomers with the respective S configuration. NMR spectra of individual diastereomers of dACA were studied, revealing characteristic differences in chemical shifts which may prove useful in configurational assignments of longer oligomers. Thus, these data provide a methodological basis for synthesis and configurational assignment of longer methylphosphonate oligomers to use as antisense probes.     

Canine 5S rRNA: nucleotide sequence and chromosomal assignment of its gene cluster in four canid species

The purpose of this study was to determine the nucleotide sequence of canine 5S rRNA and use this information to develop a molecular probe to assign the gene locus to chromosomes of the dog and three other related canid species using fluorescence in situ hybridization. The nucleotide sequence of canine liver 5S rRNA is 120 base pairs long and identical to the 5S rRNA nucleotide sequence of all other mammalian species investigated so far. A single 5S rRNA gene cluster was localized pericentromerically on chromosomes of four canid species: dog 4q1.3, red fox 4q1.3, blue fox 3q1.3 and Chinese raccoon dog 8q1.3. Chromosome arms carrying the 5S rRNA gene cluster showed striking similarities in their QFQ banding patterns, suggesting high conservation of these chromosome arms among the four species studied. The chromosomal assignments of 5S rRNA genes are among the first gene mapping results for the blue fox and the Chinese raccoon dog, and are in accordance with published data on comparative chromosome maps from human, dog, red fox, blue fox and raccoon dogs.     

Structure of DNA hexamer sequence d-CGATCG by two-dimensional nuclear magnetic resonance spectroscopy and restrained molecular dynamics

Solution conformation of self-complementary DNA duplex d-CGATCG, containing 5' d-CpG 3' site for intercalation of anticancer drug, daunomycin and adriamycin, has been investigated by nuclear magnetic resonance (NMR) spectroscopy. Complete resonance assignments of all the protons (except some H5'/H5" protons) have been obtained following standard procedures based on double quantum filtered correlation spectroscopy (dQF COSY) and two-dimensional nuclear Overhauser effect (NOE) spectra. Analysis of sums of coupling constants in one-dimensional NMR spectra, cross peak patterns in dQF COSY spectra and inter proton distances shows that the DNA sequence assumes a conformation close to the B-DNA family. The deoxyribose sugar conformation is in dynamic equilibrium with predominantly S-type conformer and a minor N-type conformer with N<-->S equilibrium varying with temperature. At 325 K, the mole fraction of the N-conformer increases for some of the residues by approximately 9%. Using a total of 10 spin-spin coupling constants and 112 NOE intensities, structural refinement has been carried out using Restrained Molecular Dynamics (rMD) with different starting structures, potential functions and rMD protocols. It is observed that pseudorotation phase angle of deoxyribose sugar for A3 and T4 residues is approximately 180 degrees and approximately 120 degrees, respectively while all other residues are close to C2'endo-conformation. A large propeller twist (approximately -18 degrees) and smallest twist angle (approximately 31 degrees) at A3pT4 step, in the middle of the sequence, a wider (12 A) and shallower (3.0 A) major groove with glycosidic bond rotation as high anti at both the ends of hexanucleotide are observed. The structure shows base-sequence dependent variations and hence strong local structural heterogeneity, which may have implications in ligand binding.     

ATR-FTIR spectroscopy studies of iron-sulfur protein and cytochrome c1 in the Rhodobacter capsulatus cytochrome bc1 complex

Redox transitions in the Rhodobacter capsulatus cytochrome bc(1) complex were investigated by perfusion-induced attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy combined with synchronous visible spectroscopy, in both the wild type and a cytochrome c(1) point mutant, M183K, in which the midpoint potential of heme was lowered from the wild-type value of 320 mV to 60 mV. Overall redox difference spectra of the wild type and M183K mutant were essentially identical, indicating that the mutation did not cause any major structural perturbation. Spectra were compared with data on the bovine bc(1) complex, and tentative assignments of several bands could be made by comparison with available data on model compounds and crystallographic structures. The bacterial spectra showed contributions from ubiquinone that were much larger than in the bovine enzyme, arising from additional bound and adventitious ubiquinone. The M183K mutant enabled selective reduction of the iron-sulfur protein which in turn allowed the IR redox difference spectra of ISP and cytochrome c(1) to be deconvoluted at high signal/noise ratios, and features of these spectra are interpreted in light of structural and mechanistic information.     

NMR analysis and sequence of toxin II from the sea anemone Radianthus paumotensis

Toxin II from Radianthus paumotensis (RpII) has been investigated by high-resolution NMR and chemical sequencing methods. Resonance assignments have been obtained for this protein by the sequential approach. NMR assignments could not be made consistent with the previously reported primary sequence for this protein, and chemical methods have been used to determine a sequence with which the NMR data are consistent. Analysis of the 2D NOE spectra shows that the protein secondary structure is comprised of two sequences of beta-sheet, probably joined into a distorted continuous sheet, connected by turns and extended loops, without any regular alpha-helical segments. The residues previously implicated in activity in this class of proteins, D8 and R13, occur in a loop region.     

Two-dimensional 1H NMR of two chemically modified analogs of the basic pancreatic trypsin inhibitor. Sequence-specific resonance assignments and sequence location of conformation changes relative to the native protein

Two-dimensional nuclear magnetic resonance was used to obtain sequence specific assignments for the 1H NMR spectra of two chemically modified analogs of the basic pancreatic trypsin inhibitor. In one analog the disulfide bond 14-38 was cleaved, in the second derivative the N-terminus was transaminated. From measurements of the chemical shifts and determination of the sequence locations of slowly exchanging backbone amide protons it was found that conformational differences between the native inhibitor and the chemical modifications occur exclusively near the modification sites and that the internal hydrogen bonds are nearly fully preserved. Intriguing conformation differences with respect to the native protein are that for five residues in the transaminated inhibitor and for one residue in the reduced inhibitor multiple local conformers are indicated, and that the four internal water molecules observed in the crystal structure of the native inhibitor appear not to be preserved after reduction of the disulfide bond 14-38.