C. Yu

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.

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 glyeine 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.

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.

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.

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.

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.

Fusion induced aggregation of model vesicles studied by dynamic and static light scattering

Membrane fusion is a key step in the virus mediated cell fusion. The vesicular dispersion serves as a model system to study the membrane fusion. We employed dynamic and static light scattering to study the fusion of phosphatidylcholine vesicles in the presence of model fusion peptide fragments from the hemagglutinin HA2 protein. The fusion-induced aggregation under the present experimental setup exhibited strong pH dependence, similar to the parental viral protein. Replacement of the glycine residue at the extreme amino terminus by glutamic acid (G1E) abolished fusion activity. The average molecular mass and diameter of vesicular dispersion obtained from static and dynamic light scattering measurements respectively at neutral and acidic pH showed about three fold increase in acidic solution containing wild type fusion peptide. The light scattering data are consistent with lipid mixing results. The present work demonstrates the utility of light scattering as a facile means to monitor the fusion process.