Biomedical subjects

Chandravanu Dash

Publications and source records attributed to Chandravanu Dash.

10 recordsLinked to original sources

PubMed2006

Examining interactions of HIV-1 reverse transcriptase with single-stranded template nucleotides by nucleoside analog interference.

Crystallographic studies have implicated several residues of the p66 fingers subdomain of human immunodeficiency virus type-1 reverse transcriptase in contacting the single-stranded template overhang immediately ahead of the DNA polymerase catalytic center. This interaction presumably assists in inducing the appropriate geometry on the template base for efficient and accurate incorporation of the incoming dNTP. To investigate this, we introduced nucleoside analogs either individually or in tandem into the DNA template ahead of the catalytic center and investigated whether they induce pausing of the replication machinery before serving as the template base. Analogs included abasic tetrahydrofuran linkages, neutralizing methylphosphonate linkages, and conformationally locked nucleosides. In addition, several Phe-61 mutants were included in our analysis, based on previous data indicating that altering this residue affects both strand displacement synthesis and the fidelity of DNA synthesis. We demonstrate here that altering the topology of the template strand two nucleotides ahead of the catalytic center can interrupt DNA synthesis. Mutating Phe-61 to either Ala or Leu accentuates this defect, whereas replacement with an aromatic residue (Trp) allows the mutant enzyme to bypass the template analogs with relative ease.

DNA↗

PubMed2006

Analysis of HIV-1 replication block due to substitutions at F61 residue of reverse transcriptase reveals additional defects involving the RNase H function.

We reported previously that substitutions F61L, F61W, F61Y and F61A in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase affect strand displacement synthesis [T. S. Fisher, T. Darden and V. R. Prasad (2003) J. Mol. Biol., 325, 443-459]. We have now determined the effect of these mutations on HIV replication. All mutant viruses were replication defective. Measuring replication intermediates in infected cells did not reveal a specific block as all mutants displayed reduced DNA synthesis (wild-type>F61L>F61W>F61Y>F61A). Analysis of 2-LTR circle junctions revealed that F61W and F61Y mutants generated increased aberrant circle junctions. Circle junctions corresponding to F61Y included 3'-PPT insertions suggesting ribonuclease H defect. In vitro assays mimicking PPT primer generation indicated that F61L, F61W and F61Y mutant RTs were unaffected, while F61A mutant cleaved both at PPT/U3 junction and at +6 with similar efficiencies. In assays measuring cleavage at the RNA/DNA junction to remove the PPT primer, all mutants were significantly affected with F61Y and F61A being most severely impaired. Our results show that (i) replication block of most mutants is due to more than one biochemical defect; (ii) mutations in polymerase domain can affect the function of a distal domain; and (iii) virological analyses of RT mutations can yield insight into structure-function relationship that is otherwise not obvious.

Amino Acid Substitution↗

PubMed2005

Examining Ty3 polypurine tract structure and function by nucleoside analog interference.

We have combined nucleoside analog interference with chemical footprinting, thermal denaturation, NMR spectroscopy, and biochemical studies to understand recognition of the polypurine tract (PPT) primer of the Saccharomyces cerevisiae long terminal repeat-containing retrotransposon Ty3 by its cognate reverse transcriptase. Locked nucleic acid analogs, which constrain sugar ring geometry, were introduced pairwise throughout the PPT (-)-DNA template, whereas abasic tetrahydrofuran linkages, which lack the nucleobase but preserve the sugar phosphate backbone, were introduced throughout the (-)-strand DNA template and (+)-strand RNA primer. Collectively, our data suggest that both the 5'- and 3'-portions of the PPT-containing RNA/DNA hybrid are sensitive to nucleoside analog substitution, whereas the intervening region can be modified without altering cleavage specificity. These two regions most likely correspond to portions of the PPT that make close contact with the Ty3 reverse transcriptase thumb subdomain and RNase H catalytic center, respectively. Achieving a similar phenotype with nucleoside analogs that have different effects on duplex geometry reveals structural features that are important mediators of Ty3 PPT recognition. Finally, the results from introducing tetrahydrofuran lesions around the scissile PPT/unique 3'-sequence junction indicate that template nucleobase -1 is dispensable for catalysis, whereas a primer nucleobase on either side of the junction is necessary.

Base Sequence↗

PubMed2005

Illustration of HIV-1 protease folding through a molten-globule-like intermediate using an experimental model that implicates alpha-crystallin and calcium ions.

The folding of HIV-1 protease to its active form involves the coordination of structure formation and dimerization, which follows a hierarchy consisting of folding nuclei spanning from the active site, hinge region, and dimerization domain. However, the biochemical characteristics of the folding intermediates of this protein remain to be elucidated. In an experimental model, the denaturation of the tethered dimer of HIV-1 protease by guanidine hydrochloride revealed an alternative conformation resembling the molten-globule state. The molten-globule state binds to the molecular chaperone alpha-crystallin and prevents its aggregation; however, the chaperone alone failed to reconstitute HIV-1 protease into its active form. Calcium ion assisted in the release of active enzyme from the chaperone complex. Alpha-crystallin, a member of the small heat-shock protein, assists proteins to fold correctly; however, the underlying principle of signals responsible for chaperone-mediated protein folding remains enigmatic. X-ray photoelectron spectroscopy has been employed to provide the evidence of calcium binding to alpha-crystallin and to decipher the effect of calcium binding on the chaperone-mediated refolding of HIV-1 protease. On the basis of our spectroscopic data, we propose that calcium ions interact with the carboxyl groups of the surface-exposed acidic amino acids of alpha-crystallin bringing electrostatic interference, which plays a pivotal role in inducing conformational changes in the chaperone responsible for the release of the active enzyme.

Calcium↗

PubMed2004

Two modes of HIV-1 polypurine tract cleavage are affected by introducing locked nucleic acid analogs into the (-) DNA template.

Unusual base-pairing in a co-crystal of reverse transcriptase (RT) and a human immunodeficiency virus type 1 (HIV-1) polypurine tract (PPT)-containing RNA/DNA hybrid suggests local nucleic acid flexibility mediates selection of the plus-strand primer. Structural elements of HIV-1 RT potentially participating in recognition of this duplex include the thumb subdomain and the ribonuclease H (RNase H) primer grip, the latter comprising elements of the connection subdomain and RNase H domain. To investigate how stabilizing HIV-1 PPT structure influences its recognition, we modified the (-) DNA template by inserting overlapping locked nucleic acid (LNA) doublets and triplets. Modified RNA/DNA hybrids were evaluated for cleavage at the PPT/U3 junction. Altered specificity was observed when the homopolymeric dA.rU tract immediately 5' of the PPT was modified, whereas PPT/U3 cleavage was lost after substitutions in the adjacent dT.rA tract. In contrast, the "unzipped" portion of the PPT was moderately insensitive to LNA insertions. Although a portion of the dC.rG and neighboring dT.rA tract were minimally affected by LNA insertion, RNase H activity was highly sensitive to altering the junction between these structural elements. Using 3'-end-labeled PPT RNA primers, we also identified novel cleavage sites ahead (+5/+6) of the PPT/U3 junction. Differential cleavage at the PPT/U3 junction and U3 + 5/+6 site in response to LNA-induced template modification suggests two binding modes for HIV-1 RT, both of which may be controlled by the interaction of its thumb subdomain (potentially via the minor groove binding track) at either site of the unzipped region.

Base Sequence↗

PubMed2004

Using pyrrolo-deoxycytosine to probe RNA/DNA hybrids containing the human immunodeficiency virus type-1 3' polypurine tract.

Recent structural analyses indicate that localized regions of abnormal base pairing exist within RNA/DNA hybrids containing the HIV-1 polypurine tract (PPT) and that these distortions may play a role in PPT function. To examine this directly, we have introduced pyrrolo-deoxycytosine (pdC), a fluorescent, environmentally sensitive analog of deoxycytosine (dC), into the DNA strand of PPT-containing hybrids. Steady-state fluorescence analysis of these hybrids reveals that the DNA base 11 nt from the PPT-U3 junction is unpaired even in the absence of reverse transcriptase (RT). Unstable base pairing is also observed within the (rG:dC)6 tract in the downstream portion of the duplex, suggesting that HIV-1 RT may recognize multiple pre-existing distortions during PPT selection. HIV-1 RT hydrolyzes pdC-containing hybrids primarily at the PPT-U3 junction, indicating that the analog does not induce a gross structural deformation of the duplex. However, aberrant cleavage is frequently observed 3 bp from the site of pdC substitution, most likely reflecting a specific interaction between the analog and amino acid residues within the RNase H primer grip. pdC substitution within the template strand of a DNA duplex does not appear to significantly affect RT-catalyzed DNA synthesis. Implications of these findings on the use of pdC to examine nucleic acid structure are discussed.

Adenine↗

PubMed2003

Slow tight binding inhibition of proteinase K by a proteinaceous inhibitor: conformational alterations responsible for conferring irreversibility to the enzyme-inhibitor complex.

The kinetics of slow onset inhibition of Proteinase K by a proteinaceous alkaline protease inhibitor (API) from a Streptomyces sp. is presented. The kinetic analysis revealed competitive inhibition of Proteinase K by API with an IC50 value 5.5 +/- 0.5 x 10-5 m. The progress curves were time-dependent, consistent with a two-step slow tight binding inhibition. The first step involved a rapid equilibrium for formation of reversible enzyme-inhibitor complex (EI) with a Ki value 5.2 +/- 0.6 x 10-6 m. The EI complex isomerized to a stable complex (EI*) in the second step because of inhibitor-induced conformational changes, with a rate constant k5 (9.2 +/- 1 x 10-3 s-1). The rate of dissociation of EI* (k6) was slower (4.5 +/- 0.5 x 10-5 s-1) indicating the tight binding nature of the inhibitor. The overall inhibition constant Ki* for two-step inhibition of Proteinase K by API was 2.5 +/- 0.3 x 10-7 m. Time-dependent dissociation of EI* revealed that the complex failed to dissociate after a time point and formed a conformationally altered, irreversible complex EI**. These conformational states of enzyme-inhibitor complexes were characterized by fluorescence spectroscopy. Tryptophanyl fluorescence of Proteinase K was quenched as a function of API concentration without any shift in the emission maximum indicating a subtle conformational change in the enzyme, which is correlated to the isomerization of EI to EI*. Time-dependent shift in the emission maxima of EI* revealed the induction of gross conformational changes, which can be correlated to the irreversible conformationally locked EI** complex. API binds to the active site of the enzyme as demonstrated by the abolished fluorescence of 5-iodoacetamidofluorescein-labeled Proteinase K. The chemoaffinity labeling experiments lead us to hypothesize that the inactivation of Proteinase K is because of the interference in the electronic microenvironment and disruption of the hydrogen-bonding network between the catalytic triad and other residues involved in catalysis.

Binding Sites↗

PubMed2003

Aspartic peptidase inhibitors: implications in drug development.

The last decade has witnessed an effervescence of research interest in the development of potent inhibitors of various aspartic peptidases. As an enzyme family, aspartic peptidases are relatively a small group that has received enormous interest because of their significant roles in human diseases like involvement of renin in hypertension, cathepsin D in metastasis of breast cancer, beta-Secretase in Alzheimer's Disease, plasmepsins in malaria, HIV-1 peptidase in acquired immune deficiency syndrome, and secreted aspartic peptidases in candidal infections. There have been developments on clinically active inhibitors of HIV-1 peptidase, which have been licensed for the treatment of AIDS. The inhibitors of plasmepsins and renin are considered a viable therapeutic strategy for the treatment of malaria and hypertension. Relatively few inhibitors of cathepsin D have been reported, partly because of its uncertain role as a viable target for therapeutic intervention. The beta-secretase inhibitors OM99-2 and OM003 were designed based on the substrate specificity information. The present article is a comprehensive state-of-the-art review describing the aspartic peptidase inhibitors illustrating the recent developments in the area. In addition, the homologies between the reported inhibitor sequences have been analyzed. The understanding of the structure-function relationships of aspartic peptidases and inhibitors will have a direct impact on the design of new inhibitor drugs.

Amino Acid Sequence↗

PubMed2002

Slow-tight binding inhibition of xylanase by an aspartic protease inhibitor: kinetic parameters and conformational changes that determine the affinity and selectivity of the bifunctional nature of the inhibitor.

The first report of slow-tight inhibition of xylanase by a bifunctional inhibitor alkalo-thermophilic Bacillus inhibitor (ATBI), from an extremophilic Bacillus sp. is described. ATBI inhibits aspartic protease (Dash, C., and Rao, M. (2001) J. Biol. Chem., 276, 2487-2493) and xylanase (Xyl I) from a Thermomonospora sp. The steady-state kinetics revealed time-dependent competitive inhibition of Xyl I by ATBI, consistent with two-step inhibition mechanism. The inhibition followed a rapid equilibrium step to form a reversible enzyme-inhibitor complex (EI), which isomerizes to the second enzyme-inhibitor complex (EI*), which dissociated at a very slow rate. The rate constants determined for the isomerization of EI to EI*, and the dissociation of EI* were 13 +/- 1 x 10(-6) s(-1) and 5 +/- 0.5 x 10(-8) s(-1), respectively. The K(i) value for the formation of EI complex was 2.5 +/- 0.5 microm, whereas the overall inhibition constant K(i)* was 7 +/- 1 nm. The conformational changes induced in Xyl I by ATBI were monitored by fluorescence spectroscopy and the rate constants derived were in agreement with the kinetic data. Thus, the conformational alterations were correlated to the isomerization of EI to EI*. ATBI binds to the active site of the enzyme and disturbs the native interaction between the histidine and lysine, as demonstrated by the abolished isoindole fluorescence of o-phthalaldehyde (OPTA)-labeled Xyl I. Our results revealed that the inactivation of Xyl I is due to the disruption of the hydrogen-bonding network between the essential histidine and other residues involved in catalysis and a model depicting the probable interaction between ATBI or OPTA with Xyl I has been proposed.

Actinomycetales↗

PubMedDate not supplied

Improved performance of preordered fungal protease-stearic acid biocomposites: enhanced catalytic activity, reusability, and temporal stability.

In an earlier report on fungal protease (F-prot)-fatty acid biocomposite film formation [Gole et al. Anal. Chem. 2000, 72, 4301], it was observed that the biocatalytic activity of the immobilized enzyme was comparable to that of the free enzyme in solution. However, a somewhat negative aspect of the protocol was the steady loss in activity during reuse and storage of the biocomposite film. In this paper, we address the latter issues and demonstrate successful attempts toward the realization of efficient biocomposite films with enhanced biological activity, temporal stability, and excellent reusability. The improved performance of the F-prot-stearic acid biocomposite is accomplished by preordering the fatty acid film by incorporation of Pb(2+) ions into the lipid matrix prior to enzyme immobilization. The lead cation induces lamellar ordering in the lipid film and thus facilitates diffusion of the F-prot molecules into the lipid matrix and accessibility of the substrate molecules (hemoglobin, Hb) to the entrapped F-prot enzyme molecules. The preordering consequently leads to effective control of the "mass transport" problem and might be responsible for the enhanced biological activity ( approximately 36%) of the enzyme molecules in the biocomposite in comparison with the free enzyme in solution, as well the excellent reusability of the composite film. In addition to biocatalytic activity measurements, the formation and characterization of the F-prot-lead stearate biocomposite films was done by quartz crystal microgravimetry and X-ray diffraction.

Catalysis↗