1H-NMR study of endothelin, sequence-specific assignment of the spectrum and a solution structure

The solution conformation of the recently discovered bi-cyclic, 21 amino acid vasoconstrictor peptide, Endothelin I, has been examined by 1H-NMR in deuterated dimethyl sulphoxide. A full sequential assignment has been achieved. In addition, 19 long range NOEs were detected which were employed as distance constraints in molecular dynamics calculations to yield a possible solution structure for this new peptide.     

Two-dimensional NMR investigation of a bent DNA fragment: assignment of the proton resonances and preliminary structure analysis.

The resonances of all the non-exchangeable protons (except 5'H and 5"H) of d(CGAAAAATCGG) + d(CCGATTTTTCG), a putatively bent DNA duplex, have been assigned using 1H two-dimensional nuclear magnetic resonance methods. The nuclear Overhauser effect data indicate an overall B-form structure for this double-helical DNA undecamer. However, several features of the NMR data such as some unusually weak C8/C6 proton to C1' proton NOE cross-peaks, the presence of relatively intense C2H to C1'H NOE cross-peaks, and unusual chemical shifts of some 2", 2', and 1' protons suggest a substantial perturbation of the helix structure at the junctions and along the length of the tract of A residues. These structural deviations are considered in terms of models of DNA bending.     

Complete assignment of carbon signals in a stereospecific peptide via selective and single off-resonance proton decoupling experiments. Analysis of the carbon-13 nuclear magnetic resonance spectrum of alumichrome at 67.88 MHz.

Polypeptides and proteins in native conformation exhibit 13C NMR spectra which are highly nondegenerate. Assignment of resonances to carbons in particular residues is hence a prerequisite for a structural analysis of the spectroscopic data. For nonprotonated carbonyl carbons, the assignment can be achieved by selective (1H alpha)13C' 2J decoupling. Using this method, we have assigned the Orn1 and Gly2 carbonyl resonances in alumichrome at 67.9 MHz. We show that a single off-resonance experiment with the decoupling frequency centered in the aliphatic proton spectrum is sufficient to assign unequivocally all the protonated carbon resonances via analysis of the reduced 1J heteronuclear splittings. Alumichrome thus becomes the first complex polypeptide spin system whose 1H, 15N, and now 13C nuclear resonances have been fully identified to date. 13C chemical shifts and 1H--13C spin--spin couplings are discussed in terms of structural strain leading to specific orbital hybridizations and on the basis of polarization effects due to electron density shifts toward hydrogen-bonding and metal-binding sites. A number of 3J(13C--C--C--1H) coupling constants measured on selected multiplets after resolution enhancement were used to derive the x-related Karplus relationship 3J(theta) = (10.2 cos2 theta -- 1.3 cos theta + 0.2) Hz.     

Conformational characteristics of the trinucleoside diphosphate xyloA2'-5'xyloA2'-5'xyloA. A nuclear magnetic resonance and CD study.

In this paper the conformational analysis of the 2'-5' linked xylotrinucleotide xA2'-5'xA2'-5'xA is reported. The title compound is an analogue of A2'-5'A2'-5'A, which compound was shown to display inhibitive effects on protein synthesis. The complete 1H-NMR assignment of the high field spectral region of the xylose trimer is given. Modes of base-base stacking are extracted from coupling constant data at various temperatures. Circular dichroic (CD) spectra confirm the presence of stacked states at low temperature. Xylonucleosides are known to prefer the N-type sugar conformation. However, in the present trimer the S-type conformer is suggested to partake in stacked conformations. Two types of stacking in the two constituent dimer fragments of the trimer are proposed to rationalize the NMR data: xA(1)N-xA(2)S and xA(2)N-xA(3)S.     

A proton nuclear magnetic resonance assignment and secondary structure determination of recombinant human thioredoxin

Two-dimensional 1H NMR spectroscopy has been applied to a structural analysis of the reduced form of a recombinant human thioredoxin, a ubiquitous dithiol oxidoreductase recently isolated from an immunocompetent lymphoblastoid cell line. The sequential assignment of the spectrum, including all proline residues, has been accomplished by using experiments to demonstrate through-bond and through-space connectivities. The secondary structure has been determined by a qualitative interpretation of nuclear Overhauser effects, NH exchange data, and 3JHN alpha coupling constants. The secondary structure was found to be similar to that of the X-ray structure of Escherichia coli thioredoxin, consisting of a mixed five-stranded beta-sheet surrounded by four alpha-helices. The assignment and structural characterization of human thioredoxin was facilitated by the increased resolution and sensitivity afforded by a magnetic field strength of 600 MHz and required the use of two temperatures and two pH conditions to resolve ambiguities caused by a duplication of resonances. This duplication, extending from Phe-41 to Val-59, and including Lys-3-Ile-5, Val-24, Val-25, Asn-39, and Ile-101-Glu-103, appears to be due to heterogeneity arising from the presence or absence of the N-terminal methionine.     

Three-dimensional structure of transketolase, a thiamine diphosphate dependent enzyme, at 2.5 A resolution.

The crystal structure of Saccharomyces cerevisiae transketolase, a thiamine diphosphate dependent enzyme, has been determined to 2.5 A resolution. The enzyme is a dimer with the active sites located at the interface between the two identical subunits. The cofactor, vitamin B1 derived thiamine diphosphate, is bound at the interface between the two subunits. The enzyme subunit is built up of three domains of the alpha/beta type. The diphosphate moiety of thiamine diphosphate is bound to the enzyme at the carboxyl end of the parallel beta-sheet of the N-terminal domain and interacts with the protein through a Ca2+ ion. The thiazolium ring interacts with residues from both subunits, whereas the pyrimidine ring is buried in a hydrophobic pocket of the enzyme, formed by the loops at the carboxyl end of the beta-sheet in the middle domain in the second subunit. The structure analysis identifies amino acids critical for cofactor binding and provides mechanistic insights into thiamine catalysis.     

Selective catalysis of A.T base pair proton exchange in DNA complexes: imino proton NMR analysis.

Polyaromatic molecules with amino chain substituents, upon binding with DNA, selectively catalyze exchange of the A.T base pair protons with bulk water protons. The amine-catalyzed exchange is mediated by compounds which are A.T and G.C base sequence specific, intercalators, and outside binders. A mechanism for the selective exchange, involving transient opening and closing of individual A.T base pairs in the duplex, is discussed.     

Human mevalonate diphosphate decarboxylase: characterization, investigation of the mevalonate diphosphate binding site, and crystal structure

Expression in Escherichia coli of his-tagged human mevalonate diphosphate decarboxylase (hMDD) has expedited enzyme isolation, characterization, functional investigation of the mevalonate diphosphate binding site, and crystal structure determination (2.4 Å resolution). hMDD exhibits V_max = 6.1 ± 0.5 U/mg; K_m for ATP is 0.69 ± 0.07 mM and K_m for (R,S) mevalonate diphosphate is 28.9 ± 3.3 μM. Conserved polar residues predicted to be in the hMDD active site were mutated to test functional importance. R161Q exhibits a ∼1000-fold diminution in specific activity, while binding the fluorescent substrate analog, TNP-ATP, comparably to wild-type enzyme. Diphosphoglycolyl proline (K_i = 2.3 ± 0.3 uM) and 6-fluoromevalonate 5-diphosphate (K_i = 62 ± 5 nM) are competitive inhibitors with respect to mevalonate diphosphate. N17A exhibits a V_max = 0.25 ± 0.02 U/mg and a 15-fold inflation in K_m for mevalonate diphosphate. N17A’s K_i values for diphosphoglycolyl proline and fluoromevalonate diphosphate are inflated (>70-fold and 40-fold, respectively) in comparison with wild-type enzyme. hMDD structure indicates the proximity (2.8 Å) between R161 and N17, which are located in an interior pocket of the active site cleft. The data suggest the functional importance of R161 and N17 in the binding and orientation of mevalonate diphosphate.     

Circular dichroism as a reliable tool for anomeric assignment of glycosyl-2-phenyl-2H-1,2,3-triazole C-nucleoside analogs. A rule for prediction of their anomeric configuration

The circular dichroism (CD) of a series of acyclic C-nucleoside analogs; 4-(pentahydroxypentyl-1-yl)-2-phenyl-2H-1,2,3-triazoles [1-5] and 4-(D-glycero-D-gulo)-2-phenyl-2H-1,2,3-triazole 6, are reported. A correlation between the sign of the Cotton effect at the maximal UV absorption and the absolute configuration of the carbon atom alpha- to the triazole base moiety is reported. The CD of anomeric 4-(alpha,beta-D-arabinofuranosyl)- and 4-(alpha,beta-D-arabinopyranosyl)-2-phenyl-2H-1,2,3-triazole C-nucleosides are reported. The assignment of the anomeric configuration of C-glycosyl-2-phenyl-2H-1,2,3-triazoles from their CD spectra was found to be a simple method that relies on comparison of the sign of the Cotton effect at the maximal UV absorption and the absolute configuration of the anomeric carbon atom. A correlation between the anomeric configuration and the sign of the Cotton effect at the maximal UV absorption is deduced and generalized as a rule for prediction of the anomeric configuration of C-glycosyl-2-phenyl-2H-1,2,3-triazoles. Nuclear Overhauser effect and 13C NMR spectra supported the CD assignment rule.     

Sequential assignment of the proton NMR spectrum of isolated alpha(CO) chains from human adult hemoglobin.

In this paper we report proton two-dimensional NMR experiments on isolated alpha chains from human hemoglobin A (HbA) in the monocarboxylated state. Several J-correlated and NOE spectra in water or deuterium water and phosphate buffer (100 mM) at 310 K and pH 5.6 were acquired and analysed for the sequential assignment of the proton resonances. In addition, we used the topological data obtained from the crystal structure of alpha subunits in the monocarboxylated HbA tetramer. The assigned resonances correspond to 70% of the amino acid residues. The present results provide information on the tertiary structure of isolated alpha chains in solution, particularly in the heme region. This structure may be compared with that of the a subunits in the tetrameric HbA(CO) in crystal by comparison of observed chemical shifts and those calculated from the X-ray atomic coordinates. Overall, the global folding of the two forms are highly similar. However, this analysis points out several local conformational differences in the heme pocket and the neighboring of the unique Trp residue. Possible explanations of these differences are discussed.     

The rate, not the spectrum, of base pair substitutions changes at a GC-content transition in the human NF1 gene region: implications for the evolution of the mammalian genome structure

The human genome is composed of long stretches of DNA with distinct GC contents, called isochores or GC-content domains. A boundary between two GC-content domains in the human NF1 gene region is also a boundary between domains of early- and late-replicating sequences and of regions with high and low recombination frequencies. The perfect conservation of the GC-content distribution in this region between human and mouse demonstrates that GC-content stabilizing forces must act regionally on a fine scale at this locus. To further elucidate the nature of these forces, we report here on the spectrum of human SNPs and base pair substitutions between human and chimpanzee. The results show that the mutation rate changes exactly at the GC-content transition zone from low values in the GC-poor sequences to high values in GC-rich ones. The GC content of the GC-poor sequences can be explained by a bias in favor of GC > AT mutations, whereas the GC content of the GC-rich segment may result from a fixation bias in favor of AT > GC substitutions. This fixation bias may be explained by direct selection by the GC content or by biased gene conversion.     

Isoprenyl diphosphate synthases: protein sequence comparisons, a phylogenetic tree, and predictions of secondary structure.

Isoprenyl diphosphate synthases are ubiquitous enzymes that catalyze the basic chain-elongation reaction in the isoprene biosynthetic pathway. Pairwise sequence comparisons were made for 6 farnesyl diphosphate synthases, 6 geranylgeranyl diphosphate synthases, and a hexaprenyl diphosphate synthase. Five regions with highly conserved residues, two of which contain aspartate-rich DDXX(XX)D motifs found in many prenyltransferases, were identified. A consensus secondary structure for the group, consisting mostly of alpha-helices, was predicted for the multiply aligned sequences from amino acid compositions, computer assignments of local structure, and hydropathy indices. Progressive sequence alignments suggest that the 13 isoprenyl diphosphate synthases evolved from a common ancestor into 3 distinct clusters. The most distant separation is between yeast hexaprenyl diphosphate synthetase and the other enzymes. Except for the chromoplastic geranylgeranyl diphosphate synthase from Capsicum annuum, the remaining farnesyl and geranylgeranyl diphosphate synthases segregate into prokaryotic/archaebacterial and eukaryotic families.     

A 300 MHz and 600 MHz proton NMR study of a 12 base pair restriction fragment: investigation of structure by relaxation measurements.

The 1H NMR spectrum of a 12 base pair DNA restriction fragment has been measured at 300 and 600 MHz and resonances from over 70 protons are individually resolved. Relaxation rate measurements have been carried out at 300 MHz and compared with the theoretical predictions obtained using an isotropic rigid rotor model with coordinates derived from a Dreiding model of DNA. The model gives results that are in excellent agreement with experiment for most protons when a 7 nsec rotational correlation time is used, although agreement is improved for certain base protons by using a shorter correlation time for the sugar group, or by increasing the sugar-base interproton distances. A comparison of non-selective and selective spin-lattice relaxation rates for carbon bound protons indicates that there is extensive spin diffusion even in this short DNA fragment. Examination of the spin-spin relaxation rates for the same type of proton on different base pairs reveals little sequence effect on conformation.     

Assignment of the proton NMR spectrum of reduced and oxidized thioredoxin: sequence-specific assignments, secondary structure, and global fold

Complete proton assignments are reported for the 1H nuclear magnetic resonance (NMR) spectrum of Escherichia coli thioredoxin in the oxidized (with active-site disulfide bridge) and reduced (with two sulfhydryl groups) states. The assignments were obtained by using an integrated assignment strategy in which spin systems were identified from a combination of relayed and multiple quantum NMR techniques prior to sequential assignment. Elements of secondary structure were identified in each protein from characteristic nuclear Overhauser effects (NOE), coupling constants, and slowly exchanging amide protons. In both oxidized and reduced thioredoxin, approximately 33% of the 108 amino acid residues participate in a beta-sheet containing four major strands (three antiparallel and one parallel). A further short beta-strand is connected in a parallel fashion at the N-terminal end of the sheet. Two of the antiparallel beta-strands are connected by a 7-residue beta-bulge loop. Three helical segments, also containing approximately 33% of the amino acid residues, are well-defined in both oxidized and reduced thioredoxin. The remaining third of the molecule apparently consists of reverse turns and loops with little defined secondary structure. The global folds of oxidized and reduced thioredoxin are shown to be essentially identical. Both NOE connectivities and chemical shift values for the two proteins are very similar, except in the immediate vicinity of the active site where significant variations in the chemical shift indicate subtle conformational changes. While the overall fold of oxidized thioredoxin is the same in solution and in the crystalline state, some small differences in local conformation are apparent.     

Conformational analysis of the trinucleoside diphosphate 3'd(A2'-5'A2'-5'A). An NMR and CD study.

A 500 MHz and 300 MHz NMR study of the trinucleoside diphosphate 3'd(A2'-5'A2'-5'A) is presented. In addition, circular dichroism is used to study base stacking in the title compound. The complete 1H-NMR spectral assignment of the sugar ring proton signals is given. Information about the sugar ring (N- or S-type conformation) and about the backbone geometry along C4'-C5' and C5'-O5' bonds is obtained from the NMR coupling constants. It is shown that the trimer mainly occurs in the N-N-N stacked state at low temperatures; the presence of a minor amount of N-N-S conformational sequence is indicated.