PubMed Health⌕ Search

Biomedical subjects

W G Wu

Publications and source records attributed to W G Wu.

At least 19 recordsLinked to original sources
PubMed1997

Crystal structure of cardiotoxin V from Taiwan cobra venom: pH-dependent conformational change and a novel membrane-binding motif identified in the three-finger loops of P-type cardiotoxin.

The crystal structure of cardiotoxin V from Taiwan cobra venom (CTX A5) has been solved at pH 8.5 and refined to an R-factor of 20.7% for 7013 reflections [>2sigma(F)] between 8- and 2.19-A resolution. The refined model shows that CTX A5 exists as a dimer. The assembly consists of 974 non-hydrogen atoms from 124 residues and 73 water molecules. The global monomeric structure is similar to that determined by NMR at pH 3.7, characterized by a core formed by two beta-sheets connected with three-finger loops. However, local conformational differences are detected in two functionally important regions, loops I and II. A disparity between the NMR and X-ray structure of CTX A5 is detected near the tip of loop I and can be attributed to the difference in the protonation state of His4 at different pH, resulting in a reorientation of the His4 imidazole ring. A concerted motion of amino acid side chains located near His4 is detected and possibly contributes to the pH-dependent binding ability of CTX A5 to phospholipid model membranes. The second difference, detected at the tip of loop II, is due to the hydrophobic contact between CTX dimers in the crystal packing and the interaction of water molecules with amino acid residues in the loop II region of the CTX containing Pro31 (P-type CTX). This interaction forces loop II into a more rigid omega shape bridging the main chain at positions 27 and 34, contradictory to the flexible, tapering shape detected by NMR. Thus, a novel continuous hydrophobic column capable of binding to and possibly penetrating the membrane lipid bilayer is formed by the tips of the three-finger loops. In this respect, the X-ray crystal structure of CTX A5 may represent the CTX structure in the membrane-binding mode.

Amino Acid Sequence↗
PubMed1997

Membrane packing geometry of diphytanoylphosphatidylcholine is highly sensitive to hydration: phospholipid polymorphism induced by molecular rearrangement in the headgroup region.

Diphytanoylphosphatidylcholine (DPhPC) has often been used in the study of protein-lipid interaction and membrane channel activity, because of the general belief that it has high bilayer stability, low ion leakage, and fatty acyl packing comparable to that of phospholipid bilayers in the liquid-crystalline state. In this solid-state 31P and 2H NMR study, we find that the membrane packing geometry and headgroup orientation of DPhPC are highly sensitive to the temperature studied and its water content. The phosphocholine headgroup of DPhPC starts to change its orientation at a water content as high as approximately 16 water molecules per lipid, as evidenced by hydration-dependent 2H NMR study at room temperature. In addition, a temperature-induced structural transition in the headgroup orientation is detected in the temperature range of approximately 20-60 degrees C for lipids with approximately 8-11 water molecules per DPhPC. Dehydration of the lipid by one more water molecule leads to a nonlamellar, presumably cubic, phase formation. The lipid packing becomes a hexagonal phase at approximately 6 water molecules per lipid. A phase diagram of DPhPC in the temperature range of -40 degrees C to 80 degrees C is thus constructed on the basis of NMR results. The newly observed hydration-dependent DPhPC lipid polymorphism emphasizes the importance of molecular packing in the headgroup region in modulating membrane structure and protein-induced pore formation of the DPhPC bilayer.

Desiccation↗
PubMed1996

Conformational change and inactivation of membrane phospholipid-related activity of cardiotoxin V from Taiwan cobra venom at acidic pH.

The phospholipid binding activity of cardiotoxin V from Naja naja atra (CTX A5) was studied by use of Langmuir monolayers and found to exhibit pH-dependence in binding to phosphatidylcholine membrane with an apparent pKa around 6.0. Proton NMR investigation of the CTX A5 molecule in the presence of phosphatidylcholine micelles reveals a decrease in association of CTX A5 with membranes at low pH as a result of the protonation of His-4 near the membrane binding site of loop I region of CTX. The pH-dependent binding can be attributed mainly, but not solely, to the change in charge content of the CTX molecule upon His-4 protonation at the membrane/water interface. This is shown by analyzing the pH- and ionic strength dependence of binding of CTXs to phospholipid monolayers according to Gouy-Chapman theory. The protonation of the His-4 residue also results in a local conformational change in the loop I region since the chemical shifts of amide protons for the amino acid residues from Cys-3 to Thr-14 are all found to vary as a function of pH with an apparent pKa similar to that of His-4. Interestingly, the effect is relayed to other amino acid residues in the structural core of the protein such as those in C-terminal (Lys-60, Cys-61, and Asn-62) and triple-stranded antiparallel beta-sheet (Cys-22, Lys-24, Ala-25, Arg-38, and Ala-41) regions. An additional local conformational change in the molecule results around pH 5 as evidenced by circular dichroism spectroscopic studies, although this change does not affect the characteristic beta-sheet and three-finger loop structure of CTX molecule as revealed by two-dimensional NOESY 1H NMR study. The latter conformational change at acidic pH, however, completely inactivates CTX-induced aggregation/fusion activity of sphingomyelin vesicles. The results suggest that deciphering the functional sites of CTXs on the basis of structure and dynamics determined at low pH should be done with caution. Since 19 out of 44 CTX homologues with known amino acid sequence contain His-4, the effect of His-4 on the structure and function of CTX molecules is important and is discussed in terms of the diverse membrane targets of CTX subtypes. Also discussed is the pH-induced activation of snake venom proteins in the victim.

Animals↗
PubMed1996

The role of acidic amino acid residues in the structural stability of snake cardiotoxins.

We have recently shown that membrane-related activities of cardiotoxin V from Naja naja atra (CTX A5) are diminished at acidic pH although the overall beta-sheet structure of the molecule is maintained. In order to understand more about the mechanism of inactivation of CTX at acidic pH, we studied the effect of pH and denaturing reagents on the structural stability of CTX. We found, first, pH-induced structural transitions occurred in CTX A5 at two pH values as judged by the CD ellipticity around 195 nm: an increase in the beta-sheet content occurred around pH 4 and followed by a decrease, therein, around pH 2. The pKa of three acidic amino acid residues in CTX A5, i.e., Glu-17, Asp-42, and Asp-59, were determined to be 4.0, 3.2, and below 2.3, respectively, by NMR spectroscopy. The low pKa value of Asp-59 implies salt bridge formation between Lys-2 and Asp-59. Thus, electrostatic interaction may stabilize the three loop structure in addition to the hydrogen bonds between N- and C-termini of CTX molecule. Second, 2,2,2-trifluoroethanol (TFE) and guanidinium chloride (GdmHCI) were found to induce alpha-helical and random coil formation, respectively, in CTX A5 and eight other beta-sheet CTXs. Comparison of the relative potencies of TFE and GdmHCI to induce structural changes suggests that the amino acid residue located at position 17 plays a role in the structural stability. Specifically, CTXs containing negatively charged Glu-17 are least stable. It is suggested that Glu-17 may perturb the interaction between Lys-2 and Asp-59, and thus the overall stability of beta-sheet, in the presence of denaturing reagent. In conclusion, the perturbed structural stability of CTXs may partially explain the lower activity CTX exhibits at acidic pH. A structural model to account for the unfolding and refolding of CTX molecules without the breaking of disulfide bonds is also proposed.

Amino Acid Sequence↗
PubMed1996

Structure and dynamics of primary hydration shell of phosphatidylcholine bilayers at subzero temperatures.

Deuterium NMR relaxation and intensity measurements of the 2H-labeled H2O/dimyristoyl phosphatidylcholine bilayer were performed to understand the molecular origin of the freezing event of phospholipid headgroup and the structure and dynamics of unfrozen water molecules in the interbilayer space at subzero temperatures. The results suggest that about one to two water molecules associated with the phosphate group freeze during the freezing event of phospholipid headgroups, whereas about five to six waters near the trimethylammonium group behave as a water cluster and remain unfrozen at temperatures as low as -70 degrees C. In addition, temperature-dependent T1 and T2 relaxation times suggest that dynamic coupling occurs not only between the phosphate group and its bound water, but also between the methyl group and the adjacent water molecules. Based on these observations, the primary hydration shell of phosphatidylcholine headgroup at subzero temperatures is suggested to consist of two distinct regions: a clathrate-like water cluster, most likely a water pentamer, near the hydrophobic methyl group, and hydration water molecules associated with the phosphate group.

Deuterium↗
PubMed1995

A new method of superoxide free radical determination in ischemia and reperfusion injury of rat liver.

The present study was designed to utilize the chemiluminescence (CL) method for O2- analysis in ischemia and reperfusion of the whole rat liver. The results indicated that the O2- level was lower after 30 and 60 min ischemia. After 40 min reperfusion, the level of O2- increased obviously, reaching about 8.3-fold (30 min ischemia) and 9.0-fold (60 min ischemia) (P < 0.001, P < 0.01) respectively. The results also show that Chinese Traditional Medicines ligustrazine and salvia compound can scavenge O2- effectively.

Animals↗
PubMed1995

Solvent effect on phosphatidylcholine headgroup dynamics as revealed by the energetics and dynamics of two gel-state bilayer headgroup structures at subzero temperatures.

The packing and dynamics of lipid bilayers at the phosphocholine headgroup region within the temperature range of -40 to -110 degrees C have been investigated by solid-state nuclear magnetic resonance (NMR) measurements of selectively deuterium-labeled H2O/dimyristoylphosphatidylcholine (DMPC) bilayers. Two coexisting signals with 2H NMR quadrupolar, splittings of 36.1 and 9.3 (or smaller) kHz were detected from the -CD3 of choline methyl group. These two signals have been assigned to two coexisting gel-state headgroup structures with fast rotational motion of -CD3 and -N(CD3)3 group, respectively, with a threefold symmetry. The largest quadrupolar splitting of the NMR signal detected from the -CD2 of C alpha and C beta methylene segment was found to be 115.2 kHz, which is 10% lower than its static value of 128.2 kHz. Thus, there are extensive motions of the entire choline group of gel-state phosphatidylcholine bilayers even at a subzero temperature of -110 degrees C. These results strongly support the previous suggestion (E. J. Dufourc, C. Mayer, J. Stohrer, G. Althoff, and G. Kothe, 1992, Biophys. J. 61:42-57) that 31P chemical shift tensor elements of DMPC determined under similar conditions are not the rigid static values. The free energy difference between the two gel-state headgroup structures was determined to be 26.3 +/- 0.9 kJ/mol for fully hydrated bilayers. Furthermore, two structures with similar free energy difference were also detected for "frozen" phosphorylcholine chloride solution in a control experiment, leading to the conclusion that the two structures may be governed solely by the energetics of fully hydrated phosphocholine headgroup. The intermolecular interactions among lipids, however, stabilize the static headgroup structure as evidenced by the apparently lower free energy difference between the two structures for partially hydrated lipid bilayers. Evidence is also presented to suggest that one of the headgroup structures with trimethylammonium group rotation, which is not compatible with the static headgroup structure in crystals, is due to the dielectric relaxation of the slowly reorienting inter bilayer water molecules near the physical edge of membrane surface. Finally, a molecular model of the hydration-induced conformational changes at the torsion angle a5 of the O-C-CN+ bond is proposed to explain the two detected coexisting headgroup structures. These results emphasize the important role of the trimethylammonium group in monitoring the structure and dynamics of the lipid headgroup.

Deuterium↗
PubMed1995

Molecular order and hydration property of amine group in phosphatidylethanolamine and its N-methyl derivatives at subzero temperatures.

The molecular order and hydration properties of the amine group in phosphatidylethanolamine and its N-methyl derivatives were studied by 2H-NMR at subzero temperatures. Three coexisting signals with 2H-NMR quadrupolar splittings of 146, 106, and 28.8 KHz were detected from the fully hydrated phosphatidylethanolamine/D2O at the lowest studied temperature of -120 degrees C by using short recycle time in the applied NMR pulse sequence. These signals have been assigned to originate from frozen D2O in the interbilayer space and the deuterated amine group, i.e., -ND, with and without threefold symmetric motions. Comparative 2H-NMR studies of phosphatidylethanolamine/D2O with different degrees of methylation over a temperature range between -40 and -120 degrees C lead to the following conclusions. First, the bond angle of -D attached to the nitrogen atom of the amine group may be determined by the 2H-NMR quadrupolar splittings, i.e., 106 and 28.8 KHz, of the two coexisting signals of the deuterated amine group and found to be 112.9 for the gel-state phosphatidylethanolamine. Second, assuming the applicability of the empirical equation for the hydrogen bond distance of N+D--O with deuteron quadrupole coupling constants and using the intermolecular hydrogen bond distance of the amine group determined in single crystals of phosphatidylethanolamine bilayers, the largest measured quadrupolar splitting (delta nu Q) of N-D in this study, i.e., 106 KHz, is close to the static value. This interpretation is also consistent with the fact that the delta nu Q value determined remains constant in the temperature range between -70 and -120 degrees C. Third, the molecular order parameter of the amine group, as calculated from the ratio of the libration-averaged and static delta nu Q value for the lipid with different degrees of methylation, suggests that the perturbation of the headgroup interaction is most significant for the final methylation step. Finally, measurement of the spectral intensity of isotropic unfrozen D2O signals in D2O/phospholipid dispersions at temperatures below the homogeneous nucleation temperature of ice formation for D2O, i.e., below -34 degrees C, suggests that the first methylation step perturbs the neighboring water most significantly. Assuming that the molecular order of the amine group and the amount of unfrozen water detected under the present experimental condition can be taken as a measure of the hydrogen-bonding ability and the extent of perturbation caused by the methyl group, respectively, the gradual methylation of the amine group perturbs the interactions of the N-methylated headgroups in a nonlinear fashion. The results provide a molecular explanation for the phase behavior of phospholipids with different degrees of methylation.

Deuterium↗
PubMed1994

Expression of glutathione S-transferase-cardiotoxin fusion protein in Escherichia coli.

We report here the construction of cardiotoxin V gene, from cobra snake venom (Naja naja atra), by chemically synthesized oligonucleotides and its expression as a glutathione S-transferase-cardiotoxin fusion protein in the inclusion bodies of Escherichia coli. The expression of cardiotoxin fusion protein in protein with a yield of about 35 mg/liter culture was confirmed by highly specific anti-peptide antibodies generated against the unique amino acid residues located at the tip of loop II of cardiotoxin V. Since the fusion protein can be easily treated by CNBr to free the toxin moiety, as revealed by immunoblotting of the cleaved protein, the results provide an avenue for future structural and functional studies of cardiotoxin molecules.

Amino Acid Sequence↗
PubMed1993

Solution structure of cardiotoxin V from Naja naja atra.

Cardiotoxins are small proteins that are found in the venoms of snakes from the Elapidae family. These toxins are known to bind to and disrupt the organization, integrity, and function of the cell membrane. Most of the well-studied cardiotoxins cause depolarization of membrane potentials and/or lysis of red cells. In contrast, CTX V from Naja naja atra displays poor hemolytic activity but is proficient at inducing aggregation and fusion of sphingomyelin vesicles [Chien et al. (1991) J. Biol. Chem. 266, 3252-3259]. To determine whether the unique activity of this CTX is attributable to its tertiary structure, the solution structure of CTX V was determined by NMR methods. On the basis of these studies, this cardiotoxin has the same general topology as other members of the family, and thus its unusual properties do not arise from any gross structural differences that are detectable by solution NMR methods. Molecular dynamics calculations indicate that residues 36-50 show concerted fluctuations. On the basis of sequence similarity, we postulate that residues 30-34 are important in determining the specificity of cardiotoxins for fusion versus lysis of vesicles.

Amino Acid Sequence↗
PubMed1993

Aspartyl residue 10 is essential for ATPase activity of rat hsc70.

Three mutants of rat hsc70 were constructed, overexpressed in Escherichia coli, purified, and characterized. First, site-directed mutation was utilized to substitute Asn for Asp-10. The recombinant protein, hsc70(D10N), loses not only its peptide-stimulated ATPase activity but also its basal ATPase activity. The measured dissociation constants of ATP (0.3 microM) and S-peptide (5 microM) for hsc70(D10N), however, are virtually identical to those of hsc70. The intrinsic fluorescence spectra of hsc70(D10N) also remain largely unchanged. Therefore, the overall structure of the hsc70 protein is most likely intact after mutation. Second, the entire C-terminal peptide-binding domain was deleted and the resultant mutant contains only the N-terminal ATPase domain of hsc70. This recombinant protein, Nt-hsc70, is a peptide-independent ATPase. The ATPase activity at 37 degrees C of the Nt-hsc70, 270 pmol/h/micrograms of protein, is comparable to that of maximally peptide-activated hsc70. Third, the Asp-10 of Nt-hsc70 was replaced by Asn. Despite that this mutant, Nt-hsc70(D10N), is capable of binding ATP and it loses the capability to hydrolyze ATP. Taken together, these results indicate that aspartyl residue 10 of hsc70 is essential for ATP hydrolysis. Purified hsc70 and its mutants autophosphorylate in vitro at a substoichiometric level. On average, less than 1% of the hsc70 and Nt-hsc70 proteins are phosphorylated. Although the amount of phosphate incorporated into hsc70(D10N) and Nt-hsc70(D10) is reduced, a significant level of phosphorylation can still be achieved in these two site-directed mutants. Hence, autophosphorylation of hsc70 and its mutants is not correlated with their ability to hydrolyze ATP.

Adenosine Triphosphatases↗
PubMed1991

Freezing of phosphocholine headgroup in fully hydrated sphingomyelin bilayers and its effect on the dynamics of nonfreezable water at subzero temperatures.

Differential scanning calorimetry (DSC) and nuclear magnetic resonance (NMR) spectroscopy are applied to characterize the nonfreezable water molecules in fully hydrated D2O/sphingomyelin at temperatures below 0 degrees C. Upon cooling, DSC thermogram displays two thermal transitions peaked at -11 and -34 degrees C. The high-temperature exothermic transition corresponds to the freezing of the bulk D2O, and the low-temperature transition, which has not previously been reported, can be ascribed to the freezing of the phosphocholine headgroup in the lipid bilayer. The dynamics of nonfreezable water are also studied by 2H NMR T1 (spin-lattice relaxation time) and T2e (spin-spin relaxation time obtained by two pulse echo) measurements at 30.7 MHz and at temperatures down to -110 degrees C. The temperature dependence of the T1 relaxation time is characterized by a distinct minimum value of 2.1 +/- 0.1 ms at -30 degrees C. T2e is discontinuous at temperature around -70 degrees C, indicating another freezing-like event for the bound water at this temperature. Analysis of the relaxation data suggest that nonfreezable water undergoes both fast and slow motions at characteristic NMR time scales. The slow motions are affected when the lipid headgroup freezes.

Calorimetry, Differential Scanning↗
PubMed1991

Preliminary crystallographic analysis of cardiotoxin V with major fusion activity from Taiwan cobra (Naja naja atra) venom.

Crystals of a cardiotoxin from Taiwan cobra venom have been obtained by the vapor diffusion method using methyl pentanediol as precipitant. The crystals belong to the hexagonal space group P6(1)22 (or P6(5)22), with cell dimensions a = b = 47.5 A, c = 111.3 A, alpha = beta = 90 degrees and gamma = 120 degrees and diffract to a resolution of 2.2 A. There is one molecule per asymmetric unit and the solvent content is estimated to be 53%.

Cobra Cardiotoxin Proteins↗
PubMed1991

Phenylarsine oxide inhibits insulin-dependent glucose transport activity in rat soleus muscles.

Phenylarsine oxide (PAO) is known to block insulin-stimulated glucose transport activity in 3T3 L1 adipocytes at a post-receptor step. Herein, we demonstrate that, at right concentration, PAO also inhibits insulin activation of glucose uptake in rat soleus muscles but does not affect basal level of uptake. In control experiments, insulin stimulation of 2-deoxy-D-glucose uptake is about 400% of that of the control level. After PAO treatment, the stimulation reduces to 150% of the control. Since the intracellular level of ATP remains unchanged after PAO treatment, when measured by phosphorus-31 nmr spectroscopy, this inhibition is not due to depletion of ATP pool size. Moreover, PAO does not affect autophosphorylation of the insulin receptors purified from rat soleus muscles, implying that the PAO blockage of insulin-dependent glucose uptake in soleus muscles also may be post-receptor.

Adenosine Triphosphate↗
PubMed1991

Fusion of sphingomyelin vesicles induced by proteins from Taiwan cobra (Naja naja atra) venom. Interactions of zwitterionic phospholipids with cardiotoxin analogues.

Egg sphingomyelin vesicles were used to assay aggregation/fusion activities of proteins from Taiwan (Naja naja atra) venom to avoid the problem of phospholipase A2 contamination during protein purification. It led to the identification of a new cardiotoxin (CTX) analogue protein (CTX V) with major aggregation/fusion, but few hemolysis, activities. On the contrary, cardiotoxin (CTX III) induced significant hemolysis of human red blood cells but exhibited few aggregation/fusion activities. To study the structure/activity relationship of these CTX-induced processes, the amino acid sequence of CTX V was determined and its aggregation/fusion activity was compared with that of CTX III by transmission electron microscopy, quasielastic laser light scattering, differential scanning calorimetry, and fluorescence spectroscopy. The results show that the CTX-induced fusion process at temperatures slightly above that of the gel to liquid-crystalline phase transition of sphingomyelin vesicles can ultimately convert small sonicated vesicles into large fused vesicles with sizes of 1-2 microns. The abilities of CTX V to induce the leakage of sphingomyelin vesicles content and to cause the fusion of vesicles are approximately 10-fold higher than those of CTX III. Based on the CTX structures determined in the present and other studies, it is suggested that the amino acid residue X within the well conserved sequence of -Cys-Pro-X-Gly-Lys-Gln-Leu-Cys- plays a role in the interaction of CTX with lipid molecules. The lipid phase transition could further enhance the protein-lipid interaction in the process leading to the fusion of vesicles.

Amino Acid Sequence↗
PubMed1991

Mechanism of hemolysis of red blood cell mediated by ethanol.

The effects of ethanol on hemolysis of human red blood cells (RBCs) were studied at 21 +/- 1 degrees C in the saline buffer (138 mM NaCl, 6.1 mM Na2HPO4, 1.4 mM NaH2PO4, 5 mM glucose and pH 7.4). The hemolysis process for ethanol-treated RBCs was preceded by the leakage of the small cation K+ from the cells indicating the colloid-osmotic nature of lysis. Since the extent of membrane lesion increased with an increasing ethanol concentration, osmotic protection experiments by using solutes varying in size were carried out to estimate the diameter of the pore. Quantitative analysis of the data by considering the effect of molecular seiving of the protectants with different sizes indicated that ethanol induced formation of membrane pores with a diameter of approximately 13 A. There was no detectable release of membrane fragments as assayed by the acetylcholinesterase activity, but the membrane structures were significantly perturbed, presumably at the membrane cytoskeletal protein, as evidenced by the altered rheological properties of RBC in the presence of ethanol. It is suggested that the creation of membrane pores might involve in the deranged cytoskeletal network of ethanol-treated RBC.

Adult↗
PubMed1991

Effect of aging on phosphate metabolites of rat brain as revealed by the in vivo and in vitro 31P NMR measurements.

Changes of phosphate metabolism in brains of neonate, weaning and adult rats were compared using both in vivo and in vitro nuclear magnetic resonance spectra. Ratios of phosphocreatine/nucleoside triphosphate (PCr/NTP) were the same in neonatal brain in both in vivo and in vitro studies, but not in weaning and adult brains. This discrepancy may have resulted from extended cerebral hypoxia due to slowed freezing of the brain by the increased skull thickness and brain mass in the weaning and adult rats. Variations in in vitro extraction condition for this age-related study may lead to systematic errors in the adult rats. Nevertheless, the phosphomonoester/nucleoside triphosphate (PME/NTP) ratios in extracts of brain from neonatal rats were higher than those obtained in vivo. In addition, the glycerophosphorylethanolamine plus glycerophosphorylcholine/nucleoside triphosphate (GPE+GPC/NTP) ratios, which were not measurable in vivo, showed age-dependent increase in extracts of rat brain. Some of the phosphomonoester and phosphodiester molecules in rat brain may be undetectable in in vivo NMR analysis because of their interaction with cellular components. The total in vitro GPE and GPC concentration in brain from neonatal rat was estimated to be 0.34 mmole/g wet tissue.

Aging↗