Neel Sarovar Bhavesh

Oriented lamellar silk fibrous scaffolds to drive cartilage matrix orientation: Towards annulus fibrosus tissue engineering

A novel design of silk-based scaffold is developed using a custom-made winding machine, with fiber alignment resembling the anatomical criss-cross lamellar fibrous orientation features of the annulus fibrosus of the intervertebral disc. Crosslinking of silk fibroin fibers with chondroitin sulphate (CS) was introduced to impart superior biological functionality. The scaffolds, with or without CS, instructed alignment of expanded human chondrocytes and of the deposited extracellular matrix while supporting their chondrogenic redifferentiation. The presence of CS crosslinking could not induce statistically significant changes in the measured collagen or glycosaminoglycan content, but resulted in an increased construct stiffness. By offering the combined effect of cell/matrix alignment and chondrogenic support, the silk fibroin scaffolds developed with precise fiber orientation in lamellar form represent a suitable substrate for tissue engineering of the annulus fibrosus part of the intervertebral disc.

A unique Ni2+ -dependent and methylglyoxal-inducible rice glyoxalase I possesses a single active site and functions in abiotic stress response.

The glyoxalase system constitutes the major pathway for the detoxification of metabolically produced cytotoxin methylglyoxal (MG) into a non-toxic metabolite D-lactate. Glyoxalase I (GLY I) is an evolutionarily conserved metalloenzyme requiring divalent metal ions for its activity: Zn(2+) in the case of eukaryotes or Ni(2+) for enzymes of prokaryotic origin. Plant GLY I proteins are part of a multimember family; however, not much is known about their physiological function, structure and metal dependency. In this study, we report a unique GLY I (OsGLYI-11.2) from Oryza sativa (rice) that requires Ni(2+) for its activity. Its biochemical, structural and functional characterization revealed it to be a monomeric enzyme, possessing a single Ni(2+) coordination site despite containing two GLY I domains. The requirement of Ni(2+) as a cofactor by an enzyme involved in cellular detoxification suggests an essential role for this otherwise toxic heavy metal in the stress response. Intriguingly, the expression of OsGLYI-11.2 was found to be highly substrate inducible, suggesting an important mode of regulation for its cellular levels. Heterologous expression of OsGLYI-11.2 in Escherichia coli and model plant Nicotiana tabacum (tobacco) resulted in improved adaptation to various abiotic stresses caused by increased scavenging of MG, lower Na(+) /K(+) ratio and maintenance of reduced glutathione levels. Together, our results suggest interesting links between MG cellular levels, its detoxification by GLY I, and Ni(2+) - the heavy metal cofactor of OsGLYI-11.2, in relation to stress response and adaptation in plants.

Molecular Insights into Mammalian End-binding Protein Heterodimerization

Microtubule plus-end tracking proteins (+TIPs) are involved in many microtubule-based processes. End binding (EB) proteins constitute a highly conserved family of +TIPs. They play a pivotal role in regulating microtubule dynamics and in the recruitment of diverse +TIPs to growing microtubule plus ends. Here we used a combination of methods to investigate the dimerization properties of the three human EB proteins EB1, EB2, and EB3. Based on Förster resonance energy transfer, we demonstrate that the C-terminal dimerization domains of EBs (EBc) can readily exchange their chains in solution. We further document that EB1c and EB3c preferentially form heterodimers, whereas EB2c does not participate significantly in the formation of heterotypic complexes. Measurements of the reaction thermodynamics and kinetics, homology modeling, and mutagenesis provide details of the molecular determinants of homo- versus heterodimer formation of EBc domains. Fluorescence spectroscopy and nuclear magnetic resonance studies in the presence of the cytoskeleton-associated protein-glycine-rich domains of either CLIP-170 or p150glued or of a fragment derived from the adenomatous polyposis coli tumor suppressor protein show that chain exchange of EBc domains can be controlled by binding partners. Extension of these studies of the EBc domains to full-length EBs demonstrate that heterodimer formation between EB1 and EB3, but not between EB2 and the other two EBs, occurs both in vitro and in cells as revealed by live cell imaging. Together, our data provide molecular insights for rationalizing the dominant negative control by C-terminal EB domains and form a basis for understanding the functional role of heterotypic chain exchange by EBs in cells.

A novel protocol based on HN(C)N for rapid resonance assignment in (15N, 13C) labeled proteins: implications to structural genomics

A novel protocol, based on the HN(C)N experiment, has been developed for rapid assignment of backbone H(N) and (15)N resonances in ((15)N, (13)C) labeled proteins. The protocol exploits the directly observable (15)N and H(N) sequential correlations and the distinctive peak patterns in the different planes of the HN(C)N spectrum, depending upon the nature of the residues displaying the correlations. Glycines and prolines, which are responsible for the distinctive features, provide many check/start points for the sequential walks. These features enhance the speed of data analysis and render side chain assignments less crucial for the success of the assignments. The application of the protocol has been demonstrated with FK506 binding protein (FKBP, molecular mass 12 kDa).

Native and nonnative conformational preferences in the urea-unfolded state of barstar

The refolding of barstar from its urea‐unfolded state has been studied extensively using various spectroscopic probes and real‐time NMR, which provide global and residue‐specific information, respectively, about the folding process. Here, a preliminary structural characterization by NMR of barstar in 8 M urea has been carried out at pH 6.5 and 25°C. Complete backbone resonance assignments of the urea‐unfolded protein were obtained using the recently developed three‐dimensional NMR techniques of HNN and HN(C)N. The conformational propensities of the polypeptide backbone in the presence of 8 M urea have been estimated by examining deviations of secondary chemical shifts from random coil values. For some residues that belong to helices in native barstar, 13Cα and 13CO secondary shifts show positive deviations in the urea‐unfolded state, indicating that these residues have propensities toward helical conformations. These residues are, however, juxtaposed by residues that display negative deviations indicative of propensities toward extended conformations. Thus, segments that are helical in native barstar are unlikely to preferentially populate the helical conformation in the unfolded state. Similarly, residues belonging to β‐strands 1 and 2 of native barstar do not appear to show any conformational preferences in the unfolded state. On the other hand, residues belonging to the β‐strand 3 segment show weak nonnative helical conformational preferences in the unfolded state, indicating that this segment may possess a weak preference for populating a helical conformation in the unfolded state.

Effects of remote mutation on the autolysis of HIV-1 PR: X-ray and NMR investigations.

Autolysis rates of the C95M and C95M/C1095A mutants of a HIV-1 protease tethered dimer have been determined by real time NMR and it is observed that the double mutant has approximately two times higher rate. X-ray structure of the C95M/C1095A double mutant has been solved and refined to 2.1 A resolution. Comparison of the double mutant structure with that of C95M single mutant reveals that there is a shift in the position of the catalytic aspartates and the bound catalytic water. The mutation also causes a loss of hydrophobic packing near the dimerization domain of the protein. These observations demonstrate that subtle changes are adequate to cause significant changes in the rate of autolysis of the double mutant. This provides a rationale for the effects of remote mutations on the activity and drug resistance of the enzyme.

Metabolic engineering of chloroplasts for artemisinic acid biosynthesis and impact on plant growth

Chloroplasts offer high-level transgene expression and transgene containment due to maternal inheritance, and are ideal hosts for biopharmaceutical biosynthesis via multigene engineering. To exploit these advantages, we have expressed 12 enzymes in chloroplasts for the biosynthesis of artemisinic acid (precursor of artemisinin, antimalarial drug) in an alternative plant system. Integration of transgenes into the tobacco chloroplast genome via homologous recombination was confirmed by molecular analysis, and biosynthesis of artemisinic acid in plant leaf tissues was detected with the help of 13C NMR and ESI-mass spectrometry. The excess metabolic flux of isopentenyl pyrophosphate generated by an engineered mevalonate pathway was diverted for the biosynthesis of artemisinic acid. However, expression of megatransgenes impacted the growth of the transplastomic plantlets. By combining two exogenous pathways, artemisinic acid was produced in transplastomic plants, which can be improved further using better metabolic engineering strategies for commercially viable yield of desirable isoprenoid products.

Sequence-specific 1H, 13C and 15N NMR assignments of Cyclophilin A like protein from Piriformospora indica involved in salt stress tolerance

Cyclophilins are omnipresent proteins found in eukaryotes and prokaryotes, with presence in cytoplasm as well as in nucleus. Primary role of Cyclophilins is of peptidyl-prolyl cis–trans isomerase, a molecular chaperon action. Here, we report sequence-specific 1H, 13C and 15N resonance assignments for a Cyclophilin A like protein from Piriformospora indica. This protein is up-regulated during salt stress conditions.

Structure of RNA-interacting Cyclophilin A-like protein from Piriformospora indica that provides salinity-stress tolerance in plants

Soil salinity problems are widespread around the globe with increased risk of spreading over the years. The fungus Piriformospora indica, identified in Indian Thar desert, colonizes the roots of monocotyledon plants and provides resistance towards biotic as well as abiotic stress conditions. We have identified a cyclophilin A-like protein from P. indica (PiCypA), which shows higher expression levels during salinity stress. The transgenic tobacco plants overexpressing PiCypA develop osmotic tolerance and exhibit normal growth under osmotic stress conditions. The crystal structure and NMR spectroscopy of PiCypA show a canonical cyclophilin like fold exhibiting a novel RNA binding activity. The RNA binding activity of the protein and identification of the key residues involved in the RNA recognition is unique for this class of protein. Here, we demonstrate for the first time a direct evidence of countering osmotic stress tolerance in plant by genetic modification using a P. indica gene.

NMR identification of local structural preferences in HIV‐1 protease tethered heterodimer in 6 M guanidine hydrochloride

Understanding protein folding requires complete characterization of all the states of the protein present along the folding pathways. For this purpose nuclear magnetic resonance (NMR) has proved to be a very powerful technique because of the great detail it can unravel regarding the structure and dynamics of protein molecules. We report here NMR identification of local structural preferences in human immunodeficiency virus‐1 protease in the ‘unfolded state’. Analyses of the chemical shifts revealed the presence of local structural preferences many of which are native‐like, and there are also some non‐native structural elements. Three‐bond HN–Hα coupling constants that could be measured for some of the N‐terminal and C‐terminal residues are consistent with the native‐like β‐structure. Unusually shifted 15N and amide proton chemical shifts of residues adjacent to some prolines and tryptophans also indicate the presence of some structural elements. These conclusions are supported by amide proton temperature coefficients and nuclear Overhauser enhancement data. The locations of the residues exhibiting preferred structural propensities on the crystal structure of the protein, give useful insights into the folding mechanism of this protein.