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T K Kumar

Publications and source records attributed to T K Kumar.

At least 37 records · Page 2Linked to original sources
PubMed1998

The role of proline in the prevention of aggregation during protein folding in vitro.

Proline effectively inhibits protein aggregation during the refolding of bovine carbonic anhydrase. Other osmolytes used such as glycine and ethylene glycol fail to exhibit the 'aggregation-blockade' role shown by proline. Results of viscosity and ANS fluorescence (1-anilino-8-naphthalene sulphonic acid) experiments suggest that proline at high concentrations forms an ordered supramolecular assembly. Based on these results, it is proposed that proline behaves as a protein folding chaperone due to the formation of an ordered, amphipathic supramolecular assembly. To our knowledge, this is the first report wherein proline is proposed as a protein folding aid.

Anilino Naphthalenesulfonates↗
PubMed1997

Solution structure of toxin b, a long neurotoxin from the venom of the king cobra (Ophiophagus hannah).

The solution structure of toxin b, a long neurotoxin (73 amino acids and 5 disulfides) from the venom of Ophiophagus hannah (king cobra), has been determined using 1H NMR and dynamical simulated annealing techniques. The structures were calculated using 485 distance constraints and 52 dihedral angle restraints. The 21 structures that were obtained satisfy the experimental restraints and possess good nonbonded contacts. Analysis of the converged structures revealed that the protein consists of a core region from which three finger-like loops extend outwards. The regular secondary structure in toxin b includes a double and a triple stranded antiparallel beta sheet. Comparison with the solution structures of other long neurotoxins reveals that although the structure of toxin b is similar to those of previously reported long neurotoxins, clear local structural differences are observed in regions proposed to be involved in binding to the acetylcholine receptor. A positively charged cluster is found in the C-terminal tail, in Loop III, and in the tip of Loop II. This cationic cluster could be crucial for the binding of the long neurotoxins to the acetylcholine receptor.

Amino Acid Sequence↗
PubMed1997

Non-specific helix-induction in charged homopolypeptides by alcohols.

The specificity/non-specificity of helix-induction in charged homopolymers such as polylysine and polyglutamic acid, at neutral pH, by various alcohols namely 2,2,2-trifluoroethanol (TFE), methanol, ethanol and 1-propanol is studied. It is found that all the alcohols used, non-specifically induced helical conformation at high concentrations. In addition, the effect(s) of TFE on an all beta-sheet protein, such cardiotoxin analogue I (CTX I) from the Taiwan Cobra (Naja naja atra) is also studied. Evaluation of the helix propensity in the amino-acid sequence of CTX I using helix-coil algorithm, AGADIR, shows a total of 1.15% helical content in the protein. In CTX I, helical conformation is found to be induced at high concentrations of TFE (> or = 70% v/v). Interestingly, upon denaturation and reduction of disulfide bridges in CTX I, helix is found to be induced even at low concentrations of TFE (> or = 20% v/v). The results of this study hints at the possible influence of native tertiary structural interactions and disulfide bridges in the induction of helix by TFE.

1-Propanol↗
PubMed1997

Characterization of a partially structured state in an all-beta-sheet protein.

Cardiotoxin analogue III (CTX III) is a low-molecular-mass all-beta-sheet protein isolated from the Taiwan cobra (Naja naja atra) venom. A stable partially structured state similar to the "molten globule' state has been identified for CTX III in a 3% (w/v) solution of 2,2,2-trichloroacetic acid at 298 K. This stable state has been structurally characterized using a variety of techniques such as CD, 1-anilinonaphthalene-8-sulphonate fluorescence binding, Fourier transform IR and two-dimensional NMR spectroscopy techniques. Direct assignment of the homonuclear two-dimensional NMR spectra of the protein in 3% trichloroacetic acid showed that drastic structural perturbation had not taken place in the protein and that the 'intermediate' state retained a significant portion of the native secondary-structural interactions. It is found that about 65% of the native beta-sheet structural contacts are maintained in the partially structured state of CTX III in 3% trichloroacetic acid.

Circular Dichroism↗
PubMed1997

Specificity of helix-induction by 2,2,2-trifluoroethanol in polypeptides.

The specificity of helix-induction in polypeptides by 2,2,2-trifluoro ethanol (TFE) is studied using an all beta-sheet protein such as cardiotoxin analogue I (CTX I) from the Taiwan Cobra (Naja naja atra) and a homopolymer such as poly-L-lysine. It is found that alcohols including TFE can 'non-specifically' induce helix at high concentrations both in CTX I and polylysine at neutral pH. However, among the alcohols used, only TFE could transform the heat-induced beta-sheet conformation of polylysine at pH 11.5 into an alpha-helix. The beta-sheet to alpha-helix conversion in polylysine (in the beta-sheet conformation) occurs even at very low concentrations of TFE (< 5% v/v). In addition, experiments on the effect(s) of TFE on the denatured and reduced CTX I (rCTX I) indicate the helix-induction in rCTX I takes place at low TFE concentrations (< 20% v/v). The results of this study hint at the possible influence of disulfide bridges on the induction of helix by TFE.

Amino Acid Sequence↗
PubMed1997

Acetonitrile-induced conformational transitions in poly-L-lysine.

The effect of acetonitrile on the random coil, alpha-helix and beta-sheet conformations induced in poly-L-lysine is studied. It is found that acetonitrile at higher concentrations transforms the backbone of polylysine from a random coil to a helical conformation. Addition of acetonitrile to polylysine (pH 11.5) in the alpha-helix conformation, induces conformational changes in two stages. At concentrations below 60% v/v, acetonitrile stabilizes the helical conformation and at higher concentrations (> 70% v/v), it destabilizes the helix. beta-sheet-->alpha-helix-->random coil conformational transitions are found to occur when polylysine in the heat-induced conformation is titrated with acetonitrile. The possible mechanism(s) of action of acetonitrile in inducing these structural transitions is discussed.

Acetonitriles↗
PubMed1997

The mechanism of 2,2,2-trichloroacetic acid-induced protein precipitation.

The mechanism of 2,2,2-trichloroacetic acid (TCA)-induced precipitation of proteins is studied. The TCA-induced protein precipitation curves are observed to be U-shaped. It is bound that the protein-precipitate-inducing effects of TCA are due to the three chloro groups in the molecule. Using cardiotoxin III (CTX III) isolated from the Taiwan cobra (Naja naja atra), as a model protein, we attempt to understand the molecular basis for the TCA-induced effects. Employing circular dichroism, proton-deuterium exchange in conjunction with conventional 2D NMR techniques, and 1-anilino naphthalene-8-sulfonate-binding experiments, we demonstrate that CTX III is in a partially structured state similar to the 'A state' in 3% w/v TCA. It is postulated that the formation of this 'sticky' partial structured 'A state' in the TCA-induced unfolding pathway is responsible for the acid-induced protein precipitation.

Chemical Precipitation↗
PubMed1997

Snake venom cardiotoxins-structure, dynamics, function and folding.

Snake cardiotoxins are highly basic (pI > 10) small molecular weight (approximately 6.5 kDa), all beta-sheet proteins. They exhibit a broad spectrum of interesting biological activities. The secondary structural elements in these toxins include antiparallel double and triple stranded beta-sheets. The three dimensional structures of these toxins reveal an unique asymmetric distribution of the hydrophobic and hydrophilic amino acids. The 3D structures of closely related snake venom toxins such as neurotoxins and cardiotoxin-like basic proteins (CLBP) fail to show similar pattern(s) in the distribution of polar and nonpolar residues. Recently, many novel biological activities have been reported for cardiotoxins. However, to-date, there is no clear structure-function correlation(s) available for snake venom cardiotoxins. The aim of this comprehensive review is to summarize and critically evaluate the progress in research on the structure, dynamics, function and folding aspects of snake venom cardiotoxins.

Amino Acid Sequence↗
PubMed1997

Proline is a protein solubilizing solute.

The effect of proline on the prevention of trichloroacetic acid (TCA)-induced protein precipitation is studied. It is found that proline at high concentrations (> 4.0 M) completely prevents TCA-induced precipitation of hen egg white lysozyme. Other osmolytes such as ethylene glycol, glycerol and sucrose fail to prevent the TCA-induced precipitation of lysozyme. Viscosity and 1-anilino-8-naphthalene sulphonic acid binding experiments suggest that proline at high concentration forms an ordered supramolecular assembly. Proline is shown to increase the solubility of protein due to formation of such higher order assemblies. A model of the supra-molecular assembly of proline is proposed and a possible in vivo role of the increased levels of proline under water stress is discussed.

Animals↗
PubMed1996

Effect of chaotropic denaturant on the binding of 1-anilino-8-naphthalene sulfonic acid to proteins.

1-Anilino-8-naphthalene sulfonic acid (ANS), a hydrophobic dye, is widely used to monitor conformational changes occurring in proteins during their folding/unfolding. Using cardiotoxin III (whose conformation remains unperturbed even in 6 M urea) from the Taiwan Cobra (Naja naja atra) venom, it is demonstrated that chaotropic denaturant such as urea directly competes with the interaction between ANS and the protein. The results presented in this report, in our opinion, has significant implication(s) in the area of protein folding, arising out of ANS binding experiments.

Anilino Naphthalenesulfonates↗
PubMed1996

2,2,2-Trifluoroethanol induces helical conformation in an all beta-sheet protein.

The effect of 2,2,2-trifluoroethanol (TFE) on the structure of an all beta-sheet protein, cardiotoxin analogue II (CTX II), from the Taiwan cobra (Naja naja atra) is studied. Using circular dichroism studies, it is found that higher concentrations of TFE induced a structural transition from beta-sheet to alpha-helix, both in the native state (nCTX II) and in denatured but not disulfide reduced CTX II (dCTX II) samples. The beta-sheet to alpha-helix conversion is shown to be cooperative. However, in denatured and reduced CTX II (rCTX II), a TFE transforms a portion(s) of the protein backbone a random coil to an alpha-helical conformation. Based on the solution structure of CTX II and the physical property of TFE, a possible mechanism for the observed backbone structural transitions induced by TFE is discussed. The results described in this paper question the significance of the structure of the "molten globule" intermediate(s) obtained in organic solvents such as TFE.

Amino Acid Sequence↗
PubMed1996

Cloning, direct expression, and purification of a snake venom cardiotoxin in Escherichia coli.

The cardiotoxin analogue III (CTX III), isolated from the Taiwan cobra (Naja naja atra) venom, is a sixty-amino acid, all beta-sheet protein. We report the direct expression of CTX III from its synthetic gene as inclusion bodies in Escherichia coli. The yield of the expressed protein is about 40 mg/liter of the culture. CTX III trapped as inclusion bodies is dissolved and refolded by the slow refolding technique. The refolded protein is purified by reverse phase high performance liquid chromatography. The purified and refolded CTX III sample is further characterized by SDS-PAGE, circular dichroism, two-dimensional NMR spectroscopy and haemolytic activity. To our knowledge, this is the first report of the direct expression and purification of snake venom cardiotoxins.

Amino Acid Sequence↗
PubMed1996

Thermal denaturation of an all beta-sheet protein--identification of a stable partially structured intermediate at high temperature.

The thermal unfolding of an all beta-sheet protein, cardiotoxin analogue III, from the Taiwan Cobra (Naja naja atra) is studied at pH 2.0, 4.0 and 6.0. At pH 4.0, using circular dichroism and 1-anilino naphthalene-8-sulphonic acid (ANS) fluorescence binding studies, a stable partially structured intermediate is detected at 90 degrees C.

Anilino Naphthalenesulfonates↗
PubMed1996

Destabilisation of native tertiary structural interactions is linked to helix-induction by 2,2,2-trifluoroethanol in proteins.

The effect of 2,2,2-trifluoroethanol (TFE) on the structure of an all beta-sheet protein, cardiotoxin analogue 111 (CTX III) from the Taiwan cobra (Naja naja atra) is studied. It is found that high concentrations (> 80% v/v) of TFE induced a beta-sheet to alpha-helix structural transition. It is found that in denatured and reduced CTX III (rCTX III) helical conformation is induced even upon addition of low concentrations (> 10% v/v) of TFE. Using three other proteins, namely, ribonuclease A (RNase A), lysozyme and alpha-lactalbumin, it is been observed that helix-induction by TFE is intricately linked to drastic destabilization of native tertiary structural interactions in the proteins.

Circular Dichroism↗
PubMed1996

Induction of helical conformation in all beta-sheet proteins by trifluoroethanol.

The effect of 2,2,2-Trifluoroethanol (TFE) on the structure of five all beta-sheet proteins, isolated from the venom of the Taiwan cobra (Naja naja atra), is studied. In all the toxins used, it is observed that significant amount of alpha-helix is induced at higher concentrations of TFE. In all these proteins, the induction of helical conformation and disruption of the tertiary structure seem to occur simultaneously. The structural transitions induced by TFE in reduced and denatured protein appear to be different from those observed in the native protein(s). In our opinion, the findings reported herein could have significant implications on research in the area of protein folding.

Circular Dichroism↗
PubMed1996

Effect of organic acids in the prevention of aggregation on rapid refolding of proteins.

The effect of organic acids on the aggregation of protein(s) during rapid refolding is studied. Using egg white lysozyme, it is observed that acetic acid not only prevents aggregation, but also aids the protein to refold back to its native, biologically active state. In contrast, formic acid, propionic acid and butyric acid fail to exhibit this property. Using circular dichroism spectroscopy it has been found that an 'aggregation-insensitive' partially folded intermediate state is induced in 0.35M acetic acid.

Acetic Acid↗