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T K Kumar

Publications and source records attributed to T K Kumar.

At least 19 recordsLinked to original sources
PubMed2001

Thermodynamic characterization of the human acidic fibroblast growth factor: evidence for cold denaturation.

The thermodynamic parameters characterizing the conformational stability of the human acidic fibroblast growth factor (hFGF-1) have been determined by isothermal urea denaturation and thermal denaturation at fixed concentrations of urea using fluorescence and far-UV CD circular dichroism (CD) spectroscopy. The equilibrium unfolding transitions at pH 7.0 are adequately described by a two-state (native <--> unfolded state) mechanism. The stability of the protein is pH-dependent, and the protein unfolds completely below pH 3.0 (at 25 degrees C). hFGF-1 is shown to undergo a two-state transition only in a narrow pH range (pH 7.0-8.0). Under acidic (pH <6.0) and basic (pH >8.0) conditions, hFGF-1 is found to unfold noncooperatively, involving the accumulation of intermediates. The average temperature of maximum stability is determined to be 295.2 K. The heat capacity change (DeltaC(p)()) for the unfolding of hFGF-1 is estimated to be 2.1 +/- 0.5 kcal.mol(-1).K(-1). Temperature denaturation experiments in the absence and presence of urea show that hFGF-1 has a tendency to undergo cold denaturation. Two-dimensional (1)H-(15)N HSQC spectra of hFGF-1 acquired at subzero temperatures clearly show that hFGF-1 unfolds under low-temperature conditions. The significance of the noncooperative unfolding under acidic conditions and the cold denaturation process observed in hFGF-1 are discussed in detail.

Circular Dichroism↗
PubMed2001

Crumpled structure of the custom hydrophobic lytic peptide cecropin B3.

The solution structure of a custom lytic peptide, cecropin B3 (CB3), having two identical hydrophobic segments on both the N- and C-termini, was investigated by two-dimensional NMR spectroscopy. The need to determine the structure of this peptide is rooted in its specific ability to lyse lipid layers that have a high content of anionic lipid. The lytic activities of CB3 on cell membranes including cancer cells and bacteria is found to be less than cecropin B1. The results show that CB3 has four discrete segments forming alpha helical structures. The crumpled structure of CB3 provides evidence for the lysis of the lipid layer being via a pathway that differs from pore formation. The results in this study provide strong clues towards a rational design for a potent antimicrobial and antitumor peptide.

3T3 Cells↗
PubMed2000

15N NMR relaxation studies of free and ligand-bound human acidic fibroblast growth factor.

15N NMR relaxation data have been used to characterize the backbone dynamics of the human acidic fibroblast growth factor (hFGF-1) in its free and sucrose octasulfate (SOS)-bound states. (15)N longitudinal (R(1)), transverse (R(2)) relaxation rates and (1H)-(15)N steady-state nuclear Overhauser effects were obtained at 500 and 600 MHz (at 25 degrees C) for all resolved backbone amide groups using (1)H- detected two-dimensional NMR experiments. Relaxation data were fit to the extended model free dynamics for each NH group. The overall correlation time (tau(m)) for the free and SOS-bound forms were estimated to be 10.4 +/- 1.07 and 11.1 +/- 1.35 ns, respectively. Titration experiments with SOS reveals that the ligand binds specifically to the C-terminal domain of the protein in a 1:1 ratio. Binding of SOS to hFGF-1 is found to induce a subtle conformational change in the protein. Significant conformational exchange (R(ex)) is observed for several residues in the free form of the protein. However, in the SOS-bound form only three residues exhibit significant R(ex) values, suggesting that the dynamics on the micro- to millisecond time scale in the free form is coupled to the cis-trans-proline isomerization. hFGF-1 is a rigid molecule with an average generalized parameter (S(2)) value of 0.89 +/- 0.03. Upon binding to SOS, there is a marked decrease in the overall flexibility (S(2) = 0.94 +/- 0.02) of the hFGF-1 molecule. However, the segment comprising residues 103-111 shows increased flexibility in the presence of SOS. Significant correlation is found between residues that show high flexibility and the putative receptor binding sites on the protein.

Binding Sites↗
PubMed2000

Rapid staining of proteins on polyacrylamide gels and nitrocellulose membranes using a mixture of fluorescent dyes.

The present work describes a novel, fluorescence-based method for staining proteins on SDS-PAGE and membrane(s). In this method, proteins are stained using a mixed-dye (sulfo-rhodamine B and 1-anilino-8-naphthalene sulfonic acid (NH(4)(+))) solution. The mixed-dye staining protocol can detect proteins up to a concentration of 15 ng. This method is generally applicable to all proteins and is more sensitive than the conventional Coomassie blue method. The staining method is rapid and efficient. Staining-destaining of proteins using the mixed-dye protocol takes less than half an hour. Another interesting feature of the staining protocol described here is the applicability to the staining of proteins on nitrocellulose membranes.

Anilino Naphthalenesulfonates↗
PubMed2000

Identification and characterization of an equilibrium intermediate in the unfolding pathway of an all beta-barrel protein.

The guanidinium hydrochloride (GdnHCl)-induced unfolding of an all beta-sheet protein, the human acidic fibroblast growth factor (hFGF-1), is studied using a variety of biophysical techniques including multidimensional NMR spectroscopy. The unfolding of hFGF-1 in GdnHCl is shown to involve the formation of a stable equilibrium intermediate. Size exclusion chromotagraphy using fast protein liquid chromatography shows that the intermediate accumulates maximally at 0.96 m GdnHCl. 1-Anilinonapthalene 8-sulfonate binding, one-dimensional (1)H NMR, and limited proteolytic digestion experiments suggest that the intermediate has characteristics resembling a molten globule state. Chemical shift perturbation and hydrogen-deuterium exchange monitored by (1)H-(15)N heteronuclear single quantum coherence spectra reveal that profound structural changes in the intermediate state (in 0.96 m GdnHCl) occur in the C-terminal, heparin binding region of the protein molecule. Additionally, results of the stopped flow fluorescence experiments suggest that the kinetic refolding of hFGF-1 proceeds through the accumulation of an intermediate at low concentrations of the denaturant. To our knowledge, the present study is the first report wherein an equilibrium intermediate is characterized in detail in an all beta-barrel protein.

Anilino Naphthalenesulfonates↗
PubMed2000

Structural events during the refolding of an all beta-sheet protein.

The refolding kinetics of the 140-residue, all beta-sheet, human fibroblast growth factor (hFGF-1) is studied using a variety of biophysical techniques such as stopped-flow fluorescence, stopped-flow circular dichroism, and quenched-flow hydrogen exchange in conjunction with multidimensional NMR spectroscopy. Urea-induced unfolding of hFGF-1 under equilibrium conditions reveals that the protein folds via a two-state (native <--> unfolded) mechanism without the accumulation of stable intermediates. However, measurement of the unfolding and refolding rates in various concentrations of urea shows that the refolding of hFGF-1 proceeds through accumulation of kinetic intermediates. Results of the quenched-flow hydrogen exchange experiments reveal that the hydrogen bonds linking the N- and C-terminal ends are the first to form during the refolding of hFGF-1. The basic beta-trefoil framework is provided by the simultaneous formation of beta-strands I, IV, IX, and X. The other beta-strands comprising the beta-barrel structure of hFGF-1 are formed relatively slowly with time constants ranging from 4 to 13 s.

Fibroblast Growth Factor 1↗
PubMed2000

Comparison of the structural stability of two homologous toxins isolated from the Taiwan cobra (Naja naja atra) venom.

Cardiotoxin analogue III (CTX III) and cobrotoxin (CBTX) isolated from the Taiwan cobra venom (Naja naja atra) are structurally homologous, small molecular weight, all-beta-sheet proteins, cross-linked by four disulfide bonds at identical positions. The conformational stabilities of these toxins are compared based on temperature-dependent chemical shifts and amide proton exchange kinetics using two-dimensional NMR spectroscopy. The structure of CTX III is found to be significantly more stable than that of CBTX. In both the toxins, beta-strand III appears to constitute the stability core. In CTX III, the stability of the triple-stranded beta-sheet domain is observed to be markedly higher than the double-stranded beta-sheet segment. In contrast, in CBTX, both structural domains (double- and triple-stranded beta-sheet domains) appear to contribute equally to the stability of the protein. Estimation of the free energy of exchange (Delta G(ex)) of residues in CBTX and CTX III reveals that the enhanced stability of the structure of CTX III stems from the strong interactions among the beta-strands constituting the triple-stranded beta-sheet domain and also the molecular forces bridging the residues at the N- and C-terminal ends of the molecule.

Animals↗
PubMed2000

Proline inhibits aggregation during protein refolding.

The in vitro refolding of hen egg-white lysozyme is studied in the presence of various osmolytes. Proline is found to prevent aggregation during protein refolding. However, other osmolytes used in this study fail to exhibit a similar property. Experimental evidence suggests that proline inhibits protein aggregation by binding to folding intermediate(s) and trapping the folding intermediate(s) into enzymatically inactive, "aggregation-insensitive" state(s). However, elimination of proline from the refolded protein mixture results in significant recovery of the bacteriolytic activity. At higher concentrations (>1.5 M), proline is shown to form loose, higher-order molecular aggregate(s). The supramolecular assembly of proline is found to possess an amphipathic character. Formation of higher-order aggregates is believed to be crucial for proline to function as a protein folding aid. In addition to its role in osmoregulation under water stress conditions, the results of this study hint at the possibility of proline behaving as a protein folding chaperone.

Animals↗
PubMed2000

Elucidation of the solution structure of cardiotoxin analogue V from the Taiwan cobra (Naja naja atra)--identification of structural features important for the lethal action of snake venom cardiotoxins.

The aim of the present study is to understand the structural features responsible for the lethal activity of snake venom cardiotoxins. Comparison of the lethal potency of the five cardiotoxin isoforms isolated from the venom of Taiwan cobra (Naja naja atra) reveals that the lethal potency of CTX I and CTX V are about twice of that exhibited by CTX II, CTX III, and CTX IV. In the present study, the solution structure of CTX V has been determined at high resolution using multidimensional proton NMR spectroscopy and dynamical simulated annealing techniques. Comparison of the high resolution solution structures of CTX V with that of CTX IV reveals that the secondary structural elements in both the toxin isoforms consist of a triple and double-stranded antiparallel beta-sheet domains. Critical examination of the three-dimensional structure of CTX V shows that the residues at the tip of Loop III form a distinct "finger-shaped" projection comprising of nonpolar residues. The occurrence of the nonpolar "finger-shaped" projection leads to the formation of a prominent cleft between the residues located at the tip of Loops II and III. Interestingly, the occurrence of a backbone hydrogen bonding (Val27CO to Leu48NH) in CTX IV is found to distort the "finger-shaped" projection and consequently diminish the cleft formation at the tip of Loops II and III. Comparison of the solution structures and lethal potencies of other cardiotoxin isoforms isolated from the Taiwan cobra (Naja naja atra) venom shows that a strong correlation exists between the lethal potency and occurrence of the nonpolar "finger-shaped" projection at the tip of Loop III. Critical analysis of the structures of the various CTX isoforms from the Taiwan cobra suggest that the degree of exposure of the cationic charge (to the solvent) contributed by the invariant lysine residue at position 44 on the convex side of the CTX molecules could be another crucial factor governing their lethal potency.

Amino Acid Sequence↗
PubMed1999

Investigation of the structural stability of cardiotoxin analogue III from the Taiwan cobra by hydrogen-deuterium exchange kinetics.

The conformational stability of a small ( approximately 7 kDa), all beta-sheet protein, cardiotoxin analogue III (CTX III), from the venom of the Taiwan cobra has been investigated by hydrogen-deuterium (H/D) exchange using two-dimensional NMR spectroscopy. The H/D exchange kinetics of backbone amide protons in CTX III has been monitored at pD 3.6 and 6.6 (at 25 degrees C), for over 5000 h. Examination of H/D exchange kinetics in the protein showed that a number of slowly exchanging residues are in the hydrophobic core of the protein. The average protection factor of the amide protons of residues belonging to the triple-stranded beta-sheet domain is about 20 times greater than that of those in the double-stranded beta-sheet segment. The residues in the C-terminal tail of the molecule, though structureless, have been found to exhibit significant protection against H/D exchange. Comparison of the quenched-flow H/D exchange data on CTX III with those obtained in the present study reveals that the most slowly exchanging portion constitutes the folding core of the protein.

Amides↗
PubMed1999

Secondary structure formation is the earliest structural event in the refolding of an all beta-sheet protein.

The refolding kinetics of cobrotoxin (CBTX), a small-molecular-weight ( approximately 7 kDa) all beta-sheet protein, has been monitored using a variety of biophysical techniques. The secondary structure formation and hydrophobic collapse occur as distinct events during the refolding of the protein. Complete secondary structure formation occurs prior to the clustering of the hydrophobic residues. The late stage(s) of the refolding pathway of CBTX is characterized by change(s) in the local environment and optical asymmetry of the indole ring of the sole tryptophan residue. The results obtained in the present study, to our knowledge, represent the first unambiguous experimental support for the framework model of protein folding.

Cobra Neurotoxin Proteins↗
PubMed1999

Structurally homologous toxins isolated from the Taiwan cobra (Naja naja atra) differ significantly in their structural stability.

Cardiotoxin and neurotoxin analogues isolated from snake venom sources are highly homologous proteins (>50% homology) with similar three-dimensional structures but exhibit drastically different biological properties. In the present study, we compare the conformational stability of cardiotoxin analogue III (CTX III) and cobrotoxin (CBTX), a neurotoxin analogue, from the Taiwan cobra (Naja naja atra), using circular dichroism spectroscopy and hydrogen-deuterium (H/D) exchange techniques in conjunction with two-dimensional NMR methods. Contrary to expectations, it is found that CTX III and CBTX differ significantly in their structural stabilities. The three-dimensional structure of CBTX is less stable than that of CTX III. The amide protons of residues at the N- and C-terminal ends of the CTX III molecule are strongly protected against H/D exchange, implying that the terminal ends of the molecule are bridged together by significant numbers of hydrogen bonds. However, in CBTX, amide protons at the terminal ends of the molecule do not exhibit an significant protection against H/D exchange. Comparison of the protection factors of the various amide protons in CTX III and CBTX reveals that the extraordinary stability of CTX III stems from the strong network of interactions among the residues at the N- and C-terminal ends and also due to the tight and ordered packing of the nonpolar residues involved in the triple-stranded, anti-parallel, beta-sheet segment of the molecule.

Animals↗
PubMed1999

Influence of disulfide bonds on the induction of helical conformation in proteins.

The effect(s) of TFE (2,2,2-trifluoroethanol) on three different conformational states (native, denatured, and carboxymethylated) of CTX III and RNase A has been examined. Contrary to the general belief, the results of the present study reveal that TFE can induce helical conformation in a protein which has no sequence propensity to form a helix. It is found that the helix induction in TFE is intricately related to the destabilization of the tertiary structural conformation in proteins. More importantly, the disulfide bonds in proteins are found to have significant influence on the TFE-mediated helix induction. The results obtained in this study strongly suggest that information pertaining to the influence of disulfide bonds on helix induction need to be considered to improve the accuracy of secondary structure prediction algorithms.

Circular Dichroism↗
PubMed1998

Unfolding and refolding of cardiotoxin III elucidated by reversible conversion of the native and scrambled species.

Cardiotoxin analogue III (CTX III) isolated from the venom of the Taiwan Cobra (Naja naja atra) is a small molecular weight, all beta-sheet protein, cross-linked by four disulfide bridges. The unfolding and refolding mechanisms of CTX III have been examined by monitoring the reversible conversion of the native and scrambled species. It is found that, in the presence of a denaturant (urea/guanidinium hydrochloride) and a thiol catalyst, CTX III forms a mixture of scrambled species by shuffling its four native disulfide bonds. Complete unfolding of CTX III can be achieved using either 3.0-4.0 M guanidinium hydrochloride (GdmCl) or 5.0-6.0 M urea. It is observed that GdmCl is thermodynamically more potent but kinetically less efficient than urea in unfolding CTX III. The rate constants of unfolding of CTX III in 8 M urea are significantly greater than that obtained in 5.0 M GdmCl and 8.0 M GdmCl. Interestingly, upon removal of the denaturant, scrambled species of CTX III is found to refold spontaneously through dynamic reshuffling of the non-native disulfides to attain the native disulfide linkages. In addition, CTX III contains highly reactive lysines which are modified by trace amounts of cyanate contaminant which exists invariably even in high-grade urea solutions. The reactive lysines of CTX III are modified by cyanate both in the native and unfolded states of the protein. The modification is nonselective, and the modified product is found to consist of highly heterogeneous species. Surprisingly, these heterogeneous species of modified CTX III are observed to display stability and folding/unfolding properties indistinguishable from those of the native CTX III. The knowledge obtained from the present study, on the conditions to convert the scrambled species, could provide useful clues for a rational design for snake venom cardiotoxins with potential therapeutic applications.

Animals↗
PubMed1998

Events in the kinetic folding pathway of a small, all beta-sheet protein.

The folding of cardiotoxin analogue III (CTX III), a small (60 amino acids), all beta-sheet protein from the venom of the Taiwan Cobra (Naja naja atra) is here investigated. The folding kinetics is monitored by using a variety of techniques such as NMR, fluorescence, and circular dichroism spectroscopy. The folding of the protein is complete within a time scale of 200 ms. The earliest detectable event in the folding pathway of CTX III is the formation of a hydrophobic cluster, which possess strong affinity to bind to nonpolar dye such as 1-anilino-8-napthalene-sulfonic acid. Quenched-flow deuterium-hydrogen exchange experiments indicate that the segment spanning residues 51-55 along with Lys23, Ile39, Val49, Tyr51 and Val52 could constitute the "hydrophobic cluster." Folding kinetics of CTX III based on the amide-protection data reveals that the triple-stranded, antiparallel beta-sheet segment, which is located in the central core of the molecule, appears to fold faster than the double-stranded beta-sheet segment.

Amides↗
PubMed1998

Main-chain dynamics of cardiotoxin II from Taiwan cobra (Naja naja atra) as studied by carbon-13 NMR at natural abundance: delineation of the role of functionally important residues.

Cardiotoxin analogue II (CTX II) is an all beta-sheet, small molecular mass (6.8 kDa), basic protein possessing a wide array of biological properties. Nearly complete assignment of the protonated carbon resonances has been achieved by heteronuclear NMR experiments. The study shows that the correlation between the carbon-13 chemical shifts and CTX II structure is good in general, but interesting deviations are also noticed. To characterize the internal dynamics of CTX II, longitudinal, transverse relaxation rates and heteronuclear 13C{1H} NOEs were measured for alpha-carbons at natural abundance by two-dimensional NMR spectroscopy. Relaxation measurements were obtained in a 14.1 T spectrometer for 50 residues, which are evenly spread along the CTX II polypeptide chain. Except for five alpha-carbons, all data were analyzed from a simple two-parameter spectral density function using the model free approach of Lipari and Szabo. The microdynamical parameters (S2, taue, and Rex) were calculated with an overall rotational correlation time (taum) for the protein of 4.8 ns. For most residues, the alpha-carbons exhibit fast (taue < 30 ps) restricted libration motions (S2 = 0.79-0.89). The present study reveals that the functionally important residues located at the tips of the three loops are flexible, and the flexibility of residues in this region could be important in the binding of cardiotoxins to their putative "receptors" which are postulated to be located on the erythrocyte membrane. In addition, the results obtained in the present study support the earlier predictions on the relative role of the lysine residues in the erythrocyte lytic activity of cardiotoxins.

Amino Acid Sequence↗
PubMed1998

The role of acetic acid in the prevention of salt-induced aggregation of snake venom cardiotoxins.

Snake venom cardiotoxins (CTXs) exhibit a strong tendency to aggregate upon desalting and hence it is extremely difficult to prepare salt-free cardiotoxin(s). In the present study, we describe a new method for preparation of salt-free CTX based on dialysis against acetic acid. Based on experimental observation and the three dimensional solution structure of cardiotoxin analogue III from the Taiwan cobra (Naja naja atra), a molecular mechanism for the prevention of aggregation of cardiotoxins by acetic acid is discussed. In our opinion, the results obtained in the present study would pave way for elucidating the structural basis for the broad spectrum of biological activities exhibited by snake venom cardiotoxins.

Acetic Acid↗