Thallampuranam Krishnaswamy S. Kumar

kappa-Hefutoxin1, a novel toxin from the scorpion Heterometrus fulvipes with unique structure and function. Importance of the functional diad in potassium channel selectivity

An important and exciting challenge in the postgenomic era is to understand the functions of newly discovered proteins based on their structures. The main thrust is to find the common structural motifs that contribute to specific functions. Using this premise, here we report the purification, solution NMR, and functional characterization of a novel class of weak potassium channel toxins from the venom of the scorpion Heterometrus fulvipes. These toxins, κ-hefutoxin1 and κ-hefutoxin2, exhibit no homology to any known toxins. NMR studies indicate that κ-hefutoxin1 adopts a unique three-dimensional fold of two parallel helices linked by two disulfide bridges without any β−sheets. Based on the presence of the functional diad (Tyr5/Lys19) at a distance (6.0 ± 1.0 Å) comparable with other potassium channel toxins, we hypothesized its function as a potassium channel toxin. κ-Hefutoxin 1 not only blocks the voltage-gated K+-channels, Kv1.3 and Kv1.2, but also slows the activation kinetics of Kv1.3 currents, a novel feature of κ-hefutoxin 1, unlike other scorpion toxins, which are considered solely pore blockers. Alanine mutants (Y5A, K19A, and Y5A/K19A) failed to block the channels, indicating the importance of the functional diad.

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.

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.

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

The guanidinium hydrochloride (GdnHCl)-induced unfolding of an all β-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 1H 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 1H-15N 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 β-barrel protein.

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.

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 β-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 β-sheet segment, which is located in the central core of the molecule, appears to fold faster than the double-stranded β-sheet segment.

Oligomerization of acidic fibroblast growth factor is not a prerequisite for its cell proliferation activity

Oligomerization of fibroblast growth factors (FGFs) induced on binding to heparin or heparan sulfate proteoglycan is considered to be crucial for receptor activation and initiation of biological responses. To gain insight into the mechanism of activation of the receptor by FGFs, in the present study we investigate the effect(s) of interaction of a heparin analog, sucrose octasulfate (SOS), on the structure, stability, and biological activities of a recombinant acidic FGF from Notophthalmus viridescens (nFGF‐1). SOS is found to bind to nFGF‐1 and significantly increase the thermodynamic stability of the protein. Using a variety of techniques such as size‐exclusion chromatography, sedimentation velocity, and multidimensional nuclear magnetic resonance (NMR) spectroscopy, it is shown that binding of SOS to nFGF‐1 retains the protein in its monomeric state. In its monomeric state (complexed to SOS), n‐FGF‐1 shows significant cell proliferation activity. 15N and 1H chemical shift perturbation and the intermolecular nuclear Overhauser effects (NOEs) between SOS and nFGF‐1 reveal that the ligand binds to the dense, positively charged cluster located in the groove enclosed by β‐strands 10 and 11. In addition, molecular modeling based on the NOEs observed for the SOS‐nFGF‐1 complex, indicates that SOS and heparin share a common binding site on the protein. In conclusion, the results of the present study clearly show that heparin‐induced oligomerization of nFGF‐1 is not mandatory for its cell proliferation activity.

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. 15N longitudinal (R1), transverse (R2) relaxation rates and {1H}-15N steady-state nuclear Overhauser effects were obtained at 500 and 600 MHz (at 25 °C) for all resolved backbone amide groups using 1H- detected two-dimensional NMR experiments. Relaxation data were fit to the extended model free dynamics for each NH group. The overall correlation time (τ 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 (S2) value of 0.89 ± 0.03. Upon binding to SOS, there is a marked decrease in the overall flexibility (S2 = 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.

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 β 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.

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.