Biomedical subjects

Rajendra P Roy

Publications and source records attributed to Rajendra P Roy.

4 recordsLinked to original sources

PubMed2006

Alginate-chaperoned facile refolding of Chromobacterium viscosum lipase.

Urea denatured lipase from Chromobacterium viscosum lipase could be refolded by addition of alginate with high guluronic acid content. The refolded molecule could be recovered by affinity precipitation. This approach resulted in recovery of 80% (of original activity) as compared to classical dilution method which gave only 21% activity recovery. Dynamic light scattering showed that binding required about 45 min and activity data obtained from affinity precipitation experiments indicated that refolding was almost instantaneous after binding. Circular dichroism (CD) and fluorescence data showed that refolded molecule was identical to the native molecule. It also showed that refolding takes place at the binding stage and not at the precipitation stage. Preliminary studies showed that the refolding strategy worked equally well with lipases from wheat germ and porcine pancreas.

Alginates↗

PubMed2005

Histidylated lipid-modified Sendai viral envelopes mediate enhanced membrane fusion and potentiate targeted gene delivery.

Recent studies have demonstrated that covalent grafting of a single histidine residue into a twin-chain aliphatic hydrocarbon compound enhances its endosome-disrupting properties and thereby generates an excellent DNA transfection system. Significant increase in gene delivery efficiencies has thus been obtained by using endosome-disrupting multiple histidine functionalities in the molecular architecture of various cationic polymers. To take advantage of this unique feature, we have incorporated L-histidine (N,N-di-n-hexadecylamine) ethylamide (L(H)) in the membrane of hepatocyte-specific Sendai virosomes containing only the fusion protein (F-virosomes (Process for Producing a Targeted Gene (Sarkar, D. P., Ramani, K., Bora, R. S., Kumar, M., and Tyagi, S. K. (November 4, 1997) U. S. Patent 5,683,866))). Such L(H)-modified virosomal envelopes were four times more (p < 0.001) active in terms of fusion with its target cell membrane. On the other hand, the presence of L(H) in reconstituted influenza and vesicular stomatitis virus envelopes failed to enhance spike glycoprotein-induced membrane fusion with host cell membrane. Circular dichroism and limited proteolysis experiments with F-virosomes indicated that the presence of L(H) leads to conformational changes in the F protein. The molecular mechanism associated with the increased membrane fusion induced by L(H) has been addressed in the light of fusion-competent conformational change in F protein. Such enhancement of fusion resulted in a highly efficient gene delivery system specific for liver cells in culture and in whole animals.

Animals↗

PubMed2004

Linkage of interactions in sickle hemoglobin fiber assembly: inhibitory effect emanating from mutations in the AB region of the alpha-chain is annulled by a mutation at its EF corner.

The AB and GH regions of the alpha-chain are located in spatial proximity and contain a cluster of intermolecular contact residues of the sickle hemoglobin (HbS) fiber. We have examined the role of dynamics of AB/GH region on HbS polymerization through simultaneous replacement of non-contact Ala(19) and Ala(21) of the AB corner with more flexible Gly or rigid alpha-aminoisobutyric acid (Aib) residues. The polymerization behavior of HbS with Aib substitutions was similar to the native HbS. In contrast, Gly substitutions inhibited HbS polymerization. Molecular dynamics simulation studies of alpha-chains indicated that coordinated motion of AB and GH region residues present in native (Ala) as well as in Aib mutant was disrupted in the Gly mutant. The inhibitory effect due to Gly substitutions was further explored in triple mutants that included mutation of an inter-doublestrand contact (alphaAsn(78) --> His or Gln) at the EF corner. Although the inhibitory effect of Gly substitutions in the triple mutant was unaffected in the presence of alphaGln(78), His at this site almost abrogated its inhibitory potential. The polymerization studies of point mutants (alphaGln(78) --> His) indicated that the inhibitory effect due to Gly substitutions in the triple mutant was synergistically compensated for by the polymerization-enhancing activity of His(78). Similar synergistic coupling, between alphaHis(78) and an intra-double-strand contact point (alpha16) mutation located in the AB region, was also observed. Thus, two conclusions are made: (i) Gly mutations at the AB corner inhibit HbS polymerization by perturbing the dynamics of the AB/GH region, and (ii) perturbations of AB region (through changes in dynamics of the AB/GH region or abolition of a specific fiber contact site) that influence HbS polymerization do so in concert with alpha78 site at the EF corner. The overall results provide insights about the interaction-linkage between distant regions of the HbS tetramer in fiber assembly.

Bacterial Proteins↗

PubMed2002

Volume exclusion effect as a driving force for reverse proteolysis. Implications for polypeptide assemblage in a macromolecular crowded milieu.

Macromolecular crowding, in principle, should affect any reaction that is accompanied by significant reduction in excluded volume. Here we have examined the influence of crowding on reverse proteolysis. We show that proteosynthesis of a polypeptide product with an interacting folding motif such as coiled coil is facilitated in crowded media as a consequence of the volume exclusion effect. Further, we demonstrate that crowding could also effect the conversion of a noncovalent protein complex (fragment complementing protein) obtained by limited proteolysis to the native covalent form, but only if the formation of the native protein results in large compaction leading to a substantial volume exclusion effect. Subtilisin-catalyzed reformation of native triosephosphate isomerase (TIM) from multiple fragments is facilitated by crowding. However, a single nick in ribonuclease S (RNase S) could not be ligated under similar conditions. The failure of generation of RNase A from RNase S is consistent with the fact that the crystal structure of the two forms are almost superimposable, and hence no significant difference of volume exclusion exists between reactant (RNase S) and product (RNase A). In contrast, considerable compaction, and consequently large reduction in excluded volume, is attained through the assembly of a TIM barrel structure. Taken together, these results have implications for both in vitro as well as in vivo polypeptide assemblage by reverse proteolysis.

Amino Acid Sequence↗