Novel solution conformation of DNA observed in d(GAATTCGAATTC) by two-dimensional NMR spectroscopy

Resonance assignments of nonexchangeable base and sugar protons of the self-complementary dodecanucleotide d(GAATTCGAATTC) have been obtained by using the two-dimensional Fourier transform NMR methods correlated spectroscopy and nuclear Overhauser effect spectroscopy. Conformational details about the sugar pucker, the glycosidic dihedral angle, and the overall secondary structure of the molecule have been derived from the relative intensities of cross peaks in the two-dimensional NMR spectra in aqueous solution. It is observed that d(GAATTCGAATTC) assumes a novel double-helical structure. The solution conformations of the two complementary strands are identical, unlike those observed in a related sequence in the solid state. Most of the five-membered sugar rings adopt an unusual O1'-endo geometry. All the glycosidic dihedral angles are in the anti domain. The AATT segments A2-T5 and A8-T11 show better stacking compared to the rest of the molecule. These features fit into a right-handed DNA model for the above two segments, with the sugar geometries different from the conventional ones. There are important structural variations in the central TCG portion, which is known to show preferences for DNase I activity, and between G1-A2 and G7-A8, which are cleavage points in the EcoRI recognition sequence. The sugar puckers for G1 and G7 are significantly different from the rest of the molecule. Further, in the three segments mentioned above, the sugar phosphate geometry is such that the distances between protons on adjacent nucleotides are much larger than those expected for a right-handed DNA. We suggest that such crevices in the DNA structure may act as "hot points" in initiation of protein recognition.     

Sequence-specific solution structure of d-GGTACGCGTACC

Complete resonance assignments of nonexchangeable base protons and sugar protons in d-GGTACGCGTACC at 500 MHz have been obtained by two-dimensional correlated spectroscopy (COSY) and nuclear Overhauser enhancement spectroscopy (NOESY). The characteristic phase-sensitive multiplet patterns of the ntrasugar cross peaks in the omega 1-scaled COSY spectrum have been used to estimate several scalar coupling constants (J). These coupling constants combined with the intranucleotide COSY cross peak intensities have been used to identify the sugar pucker of individual nucleotide units. In most cases, the deoxyribose rings adopt a conformation close to O4'-endo. Spin-diffusion has been monitored from the buildup of the normalized volumes of NOE cross peaks in NOESY spectra as a function of mixing time. A set of 55 intranucleotide and internucleotide interproton distances have been estimated from the low mixing time NOESY spectrum (tau m = 75 ms). The estimated intranucleotide proton-proton distances have been used to determine the individual glycosidic dihedral angles of the nucleotide units which lie in the anti domain. It is observed that the molecule adopts an overall conformation close to that of the B-form although there are differences in the intricate details.     

Analysis of intrasugar interproton NOESY cross-peaks as an aid to determine sugar geometries in DNA fragments

A systematic analysis of the conformation of deoxyribofuranose rings in DNA fragments has been described using two-dimensional nuclear Overhauser effect spectroscopy (2D NOESY). The approach is based on the interpretation of the intrasugar proton-proton distances which can be estimated using a low-mixing-time pure-absorption mode w1-scaled NOESY spectrum. The experimental distances are compared with the theoretical values calculated as a function of pseudorotation phase angle (P) describing the sugar geometries. The approach can be used as a complementary aid to J couplings for establishing sugar conformations in individual nucleotide units of DNA fragments. Using this strategy on d-ACATCGATGT, we observed that individual nucleotides exhibit O4'-endo sugar pucker. The results rule out possibilities of the existence of a fast equilibrium (on the NMR time scale) between C2'-endo (or S-domain) and C3'-endo (or N-domain) sugar puckers.     

Inactivation of thiostrepton by copper(II)

Summary Among the various bivalent metal ions tested, only copper(II) was found to bind to thiostrepton (M _rr 1650) in a stoichiometric ratio of 4:1. The binding of four copper ions to a thiostrepton molecule resulted in (a) irreversible loss in biological activity and (b) a change in the ultraviolet absorption spectrum of the antibiotic. Potentiometric titration of thiostrepton in the presence of copper(II) revealed dissociation of the antibiotic with a loss of 11 protons/molecule. Based on the preferential ability of copper(II) to bind to thiostrepton in the presence of some copper-complexing compounds containing similar ligand groups to the antibiotic, the possible co-ordinating atoms of the thiostrepton molecule involved in binding to the metal ion are discussed.     

Evidence for three differentially regulated catalase genes in Neurospora crassa: effects of oxidative stress, heat shock, and development.

Genetic and biochemical studies demonstrated that Neurospora crassa possesses three catalases encoded by three separate structural genes. The specific activities of the three enzymes varied in response to superoxide-mediated stress, heat shock, and development. The three loci, which we designated cat-1, cat-2, and cat-3, map to the right arms of chromosomes III, VII, and III, respectively. The cat-1-encoded enzyme (designated Cat-1; estimated molecular weight, 315,000; pI 5.2) was the predominant catalase in rapid-growth mycelium, and its activity was substantially increased in paraquat-treated and heat-shocked mycelium. Cat-2 (Mw, 165,000; pI 5.4) was absent from rapid-growth mycelium but present at low levels in conidia and stationary-phase mycelium. It was the predominant catalase in extracts derived from mycelium that had been heat shocked for 2 h. Cat-3 (Mw, 340,000; pI 5.5) was the predominant catalase in extracts from mature conidia.     

A natively unfolded βγ-crystallin domain from Hahella chejuensis

To date, very few βγ-crystallins have been identified and structurally characterized. Several of them have been shown to bind Ca(2+) and thereby enhance their stability without any significant change in structure. Although Ca(2+)-induced conformational changes have been reported in two putative βγ-crystallins from Caulobacter crescentus and Yersinia pestis, they are shown to be partially unstructured, and whether they acquire a βγ-crystallin fold is not known. We describe here a βγ-crystallin domain, hahellin, its Ca(2+) binding properties and NMR structure. Unlike any other βγ-crystallin, hahellin is characterized as a pre-molten globule (PMG) type of natively unfolded protein domain. It undergoes drastic conformational change and acquires a typical βγ-crystallin fold upon Ca(2+) binding and hence acts as a Ca(2+)-regulated conformational switch. However, it does not bind Mg(2+). The intrinsically disordered Ca(2+)-free state and the close structural similarity of Ca(2+)-bound hahellin to a microbial βγ-crystallin homologue, Protein S, which shows Ca(2+)-dependent stress response, make it a potential candidate for the cellular functions. This study indicates the presence of a new class of natively unfolded βγ-crystallins and therefore the commencement of the possible functional roles of such proteins in this superfamily.     

Sequence specific 1H, 13C and 15N resonance assignments of hahellin in 8 M urea

The sequence specific ¹H, ¹³C and ¹⁵N resonance assignments of hahellin in 8 M urea-denatured state have been accomplished by NMR spectroscopy. Secondary chemical shift analysis reveals the native-like propensities for β-rich conformation in the denatured state.     

Structure prediction of a multi-domain EF-hand Ca2+ binding protein by PROPAINOR

PROPAINOR is a new algorithm developed for ab initio prediction of the 3D structures of proteins using knowledge-based nonparametric multivariate statistical methods. This algorithm is found to be most efficient in terms of computational simplicity and prediction accuracy for single-domain proteins as compared to other ab initio methods. In this paper, we have used the algorithm for the atomic structure prediction of a multi-domain (two-domain) calcium-binding protein, whose solution structure has been deposited in the PDB recently (PDB ID: 1JFK). We have studied the sensitivity of the predicted structure to NMR distance restraints with their incorporation as an additional input. Further, we have compared the predicted structures in both these cases with the NMR derived solution structure reported earlier. We have also validated the refined structure for proper stereochemistry and favorable packing environment with good results and elucidated the role of the central linker. Figure The predicted 3D Structure of EhCaBP with bound Ca²⁺ ions (CaBP-0). In the structure, α-helices are shown in pink and the β-strands in yellow. Ca²⁺ ions are depicted as fluorescent green balls. Some of the residues in the calcium-binding loops are depicted in space-fill representation.      

Equilibrium unfolding of neuronal calcium sensor-1: N-terminal myristoylation influences unfolding and reduces protein stiffening in the presence of calcium

Neuronal calcium sensor-1 (NCS-1), a Ca(2+)-binding protein of the calcium sensor family, modulates various functions in intracellular signaling pathways. The N-terminal glycine in this protein is myristoylated, which is presumably necessary for its physiological functions. In order to understand the structural role of myristoylation and calcium on conformational stability, we have investigated the equilibrium unfolding and refolding of myristoylated and non-myristoylated NCS-1. The unfolding of these two forms of NCS-1 in the presence of calcium is best characterized by a five-state equilibrium model, and multiple intermediates accumulate during unfolding. Calcium exerts an extrinsic stabilizing effect on both forms of the protein. In the absence of calcium, the stability of both forms is dramatically decreased, and the unfolding follows a four-state equilibrium model. The equilibrium transitions are fully reversible in the presence of calcium. Myristoylation affects the pattern of equilibrium transitions substantially but not the number of intermediates, suggesting a structural role. Our data suggest that myristoylation reduces the stiffening of the protein during initial unfolding in the presence of calcium. The effects of myristoylation are more pronounced when calcium is present, suggesting a relationship between them. Inactivating the third EF-hand motif (E120Q mutant) drastically affects the equilibrium unfolding transitions, and calcium has no effect on these transitions of the mutants. The unfolding transitions of both forms of the mutant are similar to the transitions followed by the apo forms of myristoylated and non-myristoylated NCS-1. These results suggest that the role of myristoylation in unfolding/refolding of the protein is largely dependent on the presence of calcium.