sarata sahu - Academia.edu (original) (raw)

Papers by sarata sahu

Journal of Back and Musculoskeletal Rehabilitation, Jan 22, 2008

Journal of Biomolecular NMR

Journal of Magnetic Resonance, 2000

Isotropic mixing transfer functions (T kl) in three-spin systems typical of amino acids have been... more Isotropic mixing transfer functions (T kl) in three-spin systems typical of amino acids have been analyzed in order to develop simple rules for predicting transfer maxima/minima. For certain topologies, the intrinsically complex expressions describing the transfer functions reduce to compact forms which are easy to interpret and analyze. For other topologies where compactification is not possible, an analysis of the component functions of the T kl reveals that only one or two components contribute significantly to the overall profile of the transfer function. As a result, simple rules of the thumb may be devised for reasonably accurate prediction of mixing times corresponding to local and global transfer maxima/ minima, thereby facilitating mixing time optimization in TOCSY experiments.

FEBS Letters, 1999

A calcium binding protein from Entamoeba histolytica, (EhCaBP, M r∼15 kDa) is the causative agent... more A calcium binding protein from Entamoeba histolytica, (EhCaBP, M r∼15 kDa) is the causative agent for amoebiosis and has a very low sequence homology (∼30%) with other known CaBPs. Almost complete sequence specific resonance assignments for 1H, 13C and 15N spins in EhCaBP were obtained using double and triple resonance NMR experiments. Qualitative interpretation of the nuclear Overhauser enhancements, chemical shift indices and of hydrogen exchange rates threw valuable light upon the secondary structure of this protein. CaBP is found to have two globular domains each of which consists of two pairs of helix‐loop‐helix motifs. Though this protein has a very small sequence homology with calmodulins, the topological arrangement of the α‐helices and β‐strands in EhCaBP resemble them.

Biochemistry, 2001

The following versions of software and data (see references i O) were used in the production of t... more The following versions of software and data (see references i O) were used in the production of this report:

Proteins: Structure, Function, and Genetics, 2000

Backbone dynamics of uniformly 15 N-labeled barstar have been studied at 32°C, pH 6.7, by using 1... more Backbone dynamics of uniformly 15 N-labeled barstar have been studied at 32°C, pH 6.7, by using 15 N relaxation data obtained from proton-detected 2D { 1 H}-15 N NMR spectroscopy. 15 N spin-lattice relaxation rate constants (R 1), spin-spin relaxation rate constants (R 2), and steady-state heteronuclear { 1 H}-15 N NOEs have been determined for 69 of the 86 (excluding two prolines and the Nterminal residue) backbone amide 15 N at a magnetic field strength of 14.1 Tesla. The primary relaxation data have been analyzed by using the model-free formalism of molecular dynamics, using both isotropic and axially symmetric diffusion of the molecule, to determine the overall rotational correlation time (m), the generalized order parameter (S 2), the effective correlation time for internal motions (e), and NH exchange broadening contributions (R ex) for each residue. As per the axially symmetric diffusion, the ratio of diffusion rates about the unique and perpendicular axes (D ሻ /D Ќ) is 0.82 ؎ 0.03. The two results have only marginal differences. The relaxation data have also been used to map reduced spectral densities for the NH vectors of these residues at three frequencies: 0, H , and N , where H , N are proton and nitrogen Larmor frequencies. The value of m obtained from model-free analysis of the relaxation data is 5.2 ns. The reduced spectral density analysis, however, yields a value of 5.7 ns. The m determined here is different from that calculated previously from time-resolved fluorescence data (4.1 ns). The order parameter ranges from 0.68 to 0.98, with an average value of 0.85 ؎ 0.02. A comparison of the order parameters with the X-ray B-factors for the backbone nitrogens of wild-type barstar does not show any considerable correlation. Model-free analysis of the relaxation data for seven residues required the inclusion of an exchange broadening term, the magnitude of which ranges from 2 to 9.1 s ؊1 , indicating the presence of conformational averaging motions only for a small subset of residues.

Journal of Back and Musculoskeletal Rehabilitation, Oct 6, 2009

Journal of structural and functional genomics, Jan 9, 2015

Vectors designed for protein production in Escherichia coli and by wheat germ cell-free translati... more Vectors designed for protein production in Escherichia coli and by wheat germ cell-free translation were tested using 21 well-characterized eukaryotic proteins chosen to serve as controls within the context of a structural genomics pipeline. The controls were carried through cloning, small-scale expression trials, large-scale growth or synthesis, and purification. Successfully purified proteins were also subjected to either crystallization trials or (1)H-(15)N HSQC NMR analyses. Experiments evaluated: (1) the relative efficacy of restriction/ligation and recombinational cloning systems; (2) the value of maltose-binding protein (MBP) as a solubility enhancement tag; (3) the consequences of in vivo proteolysis of the MBP fusion as an alternative to post-purification proteolysis; (4) the effect of the level of LacI repressor on the yields of protein obtained from E. coli using autoinduction; (5) the consequences of removing the His tag from proteins produced by the cell-free system; an...

Molecular and Cellular Biology, 2011

Estrogen receptor alpha (ERα), a key driver of growth in the majority of breast cancers, contains... more Estrogen receptor alpha (ERα), a key driver of growth in the majority of breast cancers, contains an unstructured transactivation domain (AF1) in its N terminus that is a convergence point for growth factor and hormonal activation. This domain is controlled by phosphorylation, but how phosphorylation impacts AF1 structure and function is unclear. We found that serine 118 (S118) phosphorylation of the ERα AF1 region in response to estrogen (agonist), tamoxifen (antagonist), and growth factors results in recruitment of the peptidyl prolyl cis/trans isomerase Pin1. Phosphorylation of S118 is critical for Pin1 binding, and mutation of S118 to alanine prevents this association. Importantly, Pin1 isomerizes the serine118-proline119 bond from a cis to trans isomer, with a concomitant increase in AF1 transcriptional activity. Pin1 overexpression promotes ligand-independent and tamoxifen-inducible activity of ERα and growth of tamoxifen-resistant breast cancer cells. Pin1 expression correlat...

Journal of Structural and Functional Genomics, 2009

The Center for Eukaryotic Structural Genomics (CESG) is a ''specialized'' or ''technology develop... more The Center for Eukaryotic Structural Genomics (CESG) is a ''specialized'' or ''technology development'' center supported by the Protein Structure Initiative (PSI). CESG's mission is to develop improved methods for the high-throughput solution of structures from eukaryotic proteins, with a very strong weighting toward human proteins of biomedical relevance. During the first three years of PSI-2, CESG selected targets representing 601 proteins from Homo sapiens, 33 from mouse, 10 from rat, 139 from Galdieria sulphuraria, 35 from Arabidopsis thaliana, 96 from Cyanidioschyzon merolae, 80 from Plasmodium falciparum, 24 from yeast, and about 25 from other eukaryotes. Notably, 30% of all structures of human proteins solved by the PSI Centers were determined at CESG. Whereas eukaryotic proteins generally are considered to be much more challenging targets than prokaryotic proteins, the technology now in place at CESG yields success rates that are comparable to those of the large production centers that work primarily on prokaryotic proteins. We describe here the technological innovations that underlie CESG's platforms for bioinformatics and laboratory information management, target selection, protein production, and structure determination by X-ray crystallography or NMR spectroscopy.

Journal of Biomolecular NMR, 2011

Perdeuteration, selective deuteration, and stereo array isotope labeling (SAIL) are valuable stra... more Perdeuteration, selective deuteration, and stereo array isotope labeling (SAIL) are valuable strategies for NMR studies of larger proteins and membrane proteins. To minimize scrambling of the label, it is best to use cell-free methods to prepare selectively labeled proteins. However, when proteins are prepared from deuterated amino acids by cell-free translation in H 2 O, exchange reactions can lead to contamination of 2 H sites by 1 H from the solvent. Examination of a sample of SAIL-chlorella ubiquitin prepared by Escherichia coli cell-free synthesis revealed that exchange had occurred at several residues (mainly at Gly, Ala, Asp, Asn, Glu, and Gln). We present results from a study aimed at identifying the exchanging sites and level of exchange and at testing a strategy for minimizing 1 H contamination during wheat germ cell-free translation of proteins produced from deuterated amino acids by adding known inhibitors of transaminases (1 mM aminooxyacetic acid) and glutamate synthetase (0.1 mM L-methionine sulfoximine). By using a wheat germ cell-free expression system, we produced [U-2 H, 15 N]-chlorella ubiquitin without and with added inhibitors, and [U-15 N]-chlorella ubiquitin as a reference to determine the extent of deuterium incorporation. We also prepared a sample of [U-13 C, 15 N]-chlorella ubiquitin, for use in assigning the sites of exchange. The added inhibitors did not reduce the protein yield and were successful in blocking hydrogen exchange at C α sites with the exception of Gly. We discovered, in addition, that partial exchange occurred with or without the inhibitors at certain side-chain methyl and methylene groups: Ala-H β , Asn-H β , Asp-H β , Gln-H γ , Glu-H γ , and Lys-H ε. The side-chain labeling pattern, in particular the mixed chiral labeling resulting from partial exchange at certain sites, should be of interest in studies of large proteins, protein complexes, and membrane proteins.

PloS one, 2012

ADAPT-NMR (Assignment-directed Data collection Algorithm utilizing a Probabilistic Toolkit in NMR... more ADAPT-NMR (Assignment-directed Data collection Algorithm utilizing a Probabilistic Toolkit in NMR) represents a groundbreaking prototype for automated protein structure determination by nuclear magnetic resonance (NMR) spectroscopy. With a [(13)C,(15)N]-labeled protein sample loaded into the NMR spectrometer, ADAPT-NMR delivers complete backbone resonance assignments and secondary structure in an optimal fashion without human intervention. ADAPT-NMR achieves this by implementing a strategy in which the goal of optimal assignment in each step determines the subsequent step by analyzing the current sum of available data. ADAPT-NMR is the first iterative and fully automated approach designed specifically for the optimal assignment of proteins with fast data collection as a byproduct of this goal. ADAPT-NMR evaluates the current spectral information, and uses a goal-directed objective function to select the optimal next data collection step(s) and then directs the NMR spectrometer to co...

Journal of Biomolecular Nmr, 2000

A novel automated approach for the sequence specific NMR assignments of 1HN, 13Ca, 13Cß, 13C'... more A novel automated approach for the sequence specific NMR assignments of 1HN, 13Ca, 13Cß, 13C'/1Ha and 15N spins in proteins, using triple resonance experimental data, is presented. The algorithm, TATAPRO (Tracked AuTomated Assignments in Proteins) utilizes the protein primary sequence and peak lists from a set of triple resonance spectra which correlate 1HN and 15N chemical shifts with those of

Journal of Molecular Biology, Feb 1, 2006

The tyrosine residues adjacent to the C termini of the hemoglobin (Hb) subunits, aY140 and bY145,... more The tyrosine residues adjacent to the C termini of the hemoglobin (Hb) subunits, aY140 and bY145, are expected to play important structural roles, because the C termini are the loci of T-state quaternary salt-bridges, and because the tyrosine side-chains bridge the H and F helices via H bonds to the aV93 and bV98 carbonyl groups. These roles have been investigated via measurements of oxygen binding, 1 H NMR spectra, resonance Raman (RR) spectra, and time-resolved resonance Raman (TR 3) spectra on site mutants in which the H/F H bonds are eliminated by replacing the tyrosine residues with phenylalanine. The TR 3 spectra confirm the hypothesis, based on TR 3 studies of wild-type Hb, that the H/F H bonds break and then reform during the sub-microsecond phase of the R-T quaternary transition. The TR 3 spectra support the inference from other mutational studies that the ab dimers act as single dynamic units in this early phase, motions of the E and F helices being coupled tightly across the dimer interface. Formation of T quaternary contacts occurs at about the same rate in the mutants as in HbA. However, these contacts are weakened substantially by the Y/F substitutions. Equilibrium perturbations are apparent also, especially for the a-subunits, in which relaxation of the Fe-His bond, strengthening of the A/E interhelical H bond, and weakening of the "switch" quaternary contact in deoxyHb are all apparent. Structural effects are less marked for the b-chain Y/F replacement, but the Bohr effect is reduced by 25%, indicating that the salt-bridge and H bond interactions of the adjacent C terminus are loosened. The a-chain replacement reduces the Bohr effect much more, consistent with the global perturbations detected by the structure probes.