Effect of salt on the urea-unfolded form of barstar probed by m value measurements

To probe for residual structure present in the urea-unfolded form of the small protein barstar, to determine how salt might modulate such structure, and to determine how such structure might affect the stability of the protein, mutant variants that display m values different from that of the wild-type protein have been studied. The mutant proteins were obtained by site-directed mutagenesis at residue positions located on the surface of the folded protein. The m value, which represents the preferential free energy of interaction of urea with the unfolded form in comparison to that with the folded state, was determined from equilibrium urea-induced unfolding curves. Mutant proteins for which the m values were significantly greater than (m(+) mutant forms), significantly smaller than (m(-) mutant forms), or similar to (m(0) mutant forms) the m value determined for the wild-type protein were studied. The unfolded forms of the m(0), m(+) and m(-) mutant proteins represent different components within the unfolded form ensemble, which differ from each other in their solvent-exposed surface areas. Hence, the m value has been used as a measure of residual structure in the unfolded form. To further understand the nature of structures present in the unfolded form ensemble, the effects of the salt KCl on the stabilities of the wild-type and the mutant proteins, as well as on the structures present in the unfolded form ensemble, were also studied. It was found that the m values of the m(0), m(+) and m(-) mutant proteins all converge to the wild-type m value in the presence of KCl. This result indicates that the salt modulates residual structure in the unfolded form by screening electrostatic interactions that maintain compact and expanded components in the unfolded protein ensemble. The use of free energy cycles has allowed the effect of salt on the structure and free energy of the unfolded protein to be related to the stability of the protein.     

Azobenzene-modified poly(l-glutamic acid) (AZOPLGA): its conformational and photodynamic properties

Azobenzene-modified poly(l-glutamic acid) (AZOPLGA) polymers with 22 and 35 mol % of azo chromophores in the side chains have been synthesized by condensing 4-methoxy-4'-aminoazobenzene and poly(l-glutamic acid). These polymers have been characterized by NMR, FT-IR, and UV-visible spectroscopic techniques. The conformational features of the polymer backbone chains in the films that were cast from the polymer solutions prepared in different solvents have been investigated by circular dichroism spectroscopy. Experimental data suggested that the thermal cis-trans relaxation and photoinduced birefringence, which are related to the azo chromophores in the side chains of polymer, are not affected by the conformations of polymer backbones. However, the modulations of the surface relief gratings, the result of photoinduced mass transport process, recorded on these polymers are sensitive to polymer main chain conformation, as well as the degree of functionalization.     

Differential salt-induced stabilization of structure in the initial folding intermediate ensemble of barstar

The effects of two salts, KCl and MgCl(2), on the stability and folding kinetics of barstar have been studied at pH 8. Equilibrium urea unfolding curves were used to show that the free energy of unfolding, deltaG(UN), of barstar increased from a value of 4.7 kcalmol(-1) in the absence of salt to a value of 6.9 kcalmol(-1) in the presence of 1M KCl or 1M MgCl(2). For both salts, deltaG(UN) increases linearly with an increase in concentration of salt from 0M to 1M, suggesting that stabilization of the native state occurs primarily through a Hofmeister effect. Refolding kinetics were studied in detail in the presence of 1M KCl as well as in the presence of 1M MgCl(2), and it is shown that the basic folding mechanism is not altered upon addition of salt. The major effects on the refolding kinetics can be attributed to the stabilization of the initial burst phase ensemble, I(E), by salt. Stabilization of structure in I(E) by KCl causes the fluorescence properties of I(E) to change, so that there is an initial burst phase change in fluorescence at 320 nm, during refolding. The structure in I(E) is stabilized by MgCl(2), but no burst phase change in fluorescence at 320 nm is observed during refolding. The fluorescence emission spectra of I(E) show that when refolding is initiated in 1M KCl, the three tryptophan residues in I(E) are less solvent exposed than when folding is initiated in 1M MgCl(2). Stabilization of I(E) leads to an acceleration in the rate of the fast observable phase of folding by both salts, suggesting that structure of the transition state resembles that of I(E). The stabilization of I(E) by salts can be accounted for largely by the same mechanism that accounts for the stabilization of the native state of the protein, namely through the Hofmeister effect. The salts do not affect the rates of the slower phases of folding, indicating that the late intermediate ensemble, I(L), is not stabilized by salts. Stabilization of the native state results in deceleration of the fast unfolding rate, which has virtually no dependence on the concentration of KCl or MgCl(2) at high concentrations. The observation that the salt-induced stabilization of structure in I(E) is accompanied by an acceleration in the fast folding rate, suggests that I(E) is likely to be a productive on-pathway intermediate.     

NMR identification and characterization of the flexible regions in the 160 kDa molten globule-like aggregate of barstar at low pH

Barstar is known to form a molten globule-like A form below pH 4. This form exists as a soluble aggregate of 16 monomeric subunits, and appears to remain homogeneous in solution for at least two weeks. Here, structural characterization by NMR of the flexible regions in the A form of barstar has been carried out at pH 2.7 and 25 degrees C. Significantly, the A form appears to be a symmetrical aggregate. Using the recently described fast assignment strategy from HNN and HN(C)N spectra, along with the standard triple resonance and three-dimensional NMR experiments, the flexible segment of the aggregate has been identified to belong largely to the N-terminal end of the polypeptide chain; sequential connectivities were obtained for the first 20 residues (except two) from these experiments. This segment is free in each of the monomeric subunits, and does not form a part of the aggregated core of the A form. The secondary chemical shifts of these residues suggest propensity toward an extended structure. Their (3)J(HN,H)(alpha) coupling constants have values corresponding to those in a random coil structure. However, a few medium-range NOEs, some of them involving side chain atoms, are observed between some residues in this segment. The lowered temperature coefficients of the H(N) chemical shifts compared to random coil values indicate possibilities of some hydrogen bonding in this region. Analysis of the (15)N relaxation parameters and reduced spectral density functions, in particular the negative values of heteronuclear NOEs, indicates large-amplitude high-frequency motions in the N-terminal segments; the first three residues show more negative NOEs than the others. The (15)N transverse relaxation rates and the J(0) spectral density values for residues Ser12 and Ser69 are significantly larger than for the rest, indicating some microsecond to millisecond time scale conformational exchange contributions to the relaxation of these residues. Taken all together, the data suggest that the A form of barstar is an aggregate with a rigid core, but with the N-terminal 20 residues of each of the monomeric subunits, in a highly dynamic random coil conformation which shows transient local ordering of structure. The N-terminal segment, anchored to the aggregated core, exhibits free-flight motion.     

Single-channel current recordings of acetylcholine receptors in electroplax isolated from theElectrophorus electricus main and Sachs' electric organs

Summary Extensive chemical kinetic measurements of acetylcholine receptor-controlled ion translocation in membrane vesicles isolated from the electroplax ofElectrophorus electricus have led to the proposal of a minimum model which accounts for the activation, desensitization, and voltage-dependent inhibition of the receptor by acetylcholine, suberyldicholine, and carbamoylcholine. Comparison of chemical kinetic measurements of the dynamic properties of the acetylcholine receptor in vesicles with the properties of the receptor in cells obtained from the same organ and animal have been hampered by an inability to make the appropriate measurements withElectrophorus electricus electroplax cells. Here we report a method for exposing and cleaning the surface of electroplax cells obtained from both the Main electric organ and the organ of Sachs and the results of single-channel current recordings which have now become possible. The single-channel current recordings were made in the presence of either carbamoylcholine or suberyldicholine, as a function of temperature and transmembrane voltage. Both the channel open times and the single-channel conductance were measured. The data were found to be consistent with the model based on chemical kinetic measurements using receptor-rich membrane vesicles prepared from the Main electric organ ofE. electricus.     

Chetomin induces apoptosis in human triple-negative breast cancer cells by promoting calcium overload and mitochondrial dysfunction

Human triple-negative breast cancer (TNBC) is poorly diagnosed and unresponsive to conventional hormone therapy. Chetomin (CHET), a fungal metabolite synthesized by Chaetomium cochliodes, has been reported as a promising anticancer and antiangiogenic agent but the complete molecular mechanism of its anticancer potential remains to be elucidated. In our study, we explored the anti-neoplastic action of CHET on TNBC cells. Cytotoxicity studies were performed in human TNBC cells viz. MDA-MB-231 and MDA-MB-468 cells by Sulforhodamine B assay. It exhibited antiproliferative response and induced apoptosis in both the cell types. Cell cycle analysis revealed that it increases the sub G0/G1 phase cell population. Modulation of mitochondrial membrane potential, activation of caspase 3/7 and a remarkable increase in the expression of cleaved PARP and increased chromatin condensation was observed after CHET treatment in MDA-MB-231 and MDA-MB-468 cells. Additionally, an elevated level of intracellular Ca²⁺ played an important role in CHET mediated cell death response. Calcium overload in mitochondria led to release of cytochrome c which in turn triggered caspase-3 mediated cell death. Inhibition of calcium signalling using BAPTA-AM reduced apoptosis confirming the involvement of calcium signalling in CHET induced cell death. Chetomin also inhibited PI3K/mTOR cell survival pathway in human TNBC cells. The overall findings suggest that Chetomin inhibited the growth of human TNBC cells by caspase-dependent apoptosis and modulation of PI3K/mTOR signalling and could be used as a novel chemotherapeutic agent for the treatment of human TNBC in future.     

Mechanistic and Structural Origins of the Asymmetric Barrier to Prion-like Cross-Seeding between Tau-3R and Tau-4R

The spread and deposition of infectious fibrillar protein aggregates in the brain via a prion-like mechanism is a critical component in the patho-physiology of various neurodegenerative diseases, including the tauopathies. In tauopathies, two isoforms of tau, containing three and four microtubule binding repeats, are found to aggregate, and the type of isoform present in aggregates determines the type of tauopathy. Cross-seeding between the two tau isoforms is limited by an asymmetric barrier similar to the species barrier that restricts prion transmission across species, whose origin has remained unclear. In this study, the growth of the tau fibrils is shown to be describable by a two-step Michaelis–Menten-like model. Delineation of the mechanism as a Michaelis–Menten-like mechanism has enabled a quantitative understanding of the asymmetric seeding barrier that exists between two isoforms of tau, tau-K18 and tau-K19 (which differ in containing four and three microtubule binding repeats, respectively), wherein tau-K18 fibrils cannot seed tau-K19 monomer. Furthermore, high-resolution structural analysis of the two isoforms shows that the structural core is more ordered in tau-K19 than in tau-K18. Hence, the current work provides kinetic and structural rationales for asymmetric seeding barriers in general and for the two tau isoforms in particular.     

2'-Deoxyadenosine Induces Apoptosis in Rat Chromaffin Cells

Abstract: We show here that 2'-deoxyadenosine (2'-dAdo) but not adenosine was toxic to chromaffin cells of 3–4-week-old rat adrenal glands. More than 75% of the cells plated in culture gradually died over a 3-day period in the presence of 100 µ M 2'-dAdo plus 3 µ M deoxycoformycin (DCF). Morphological observations together with bisbenzimide staining and terminal deoxynucleotidyl transferase-mediated nick end labeling showed membrane blebbing, shrinkage of cell bodies, chromatin condensation, and DNA fragmentation, suggesting apoptosis-like cell death by 2'-dAdo. Lethal effects of 2'-dAdo were potentiated by DCF, a drug that inhibits adenosine deaminase. 2'-dAdo-prompted cell death was not prevented by inhibitors of nucleoside transporter (3 µ M dilazep or 1 µ M nitrobenzylthioinosine), precursors of pyrimidine nucleotide biosynthesis (300 µ M uridine or 100 µ M 2'-deoxycytidine), or 5 m M nicotinamide. Cells incubated with 2'-dAdo (100 and 300 µ M ) showed a three- and ninefold, respectively, increase in content of dATP, a product known to be an inhibitor of ribonucleotide reductase, an enzyme essential for DNA synthesis. Formation of dATP was completely prevented by iodotubercidin (ITu), a drug that inhibits phosphorylation of 2'-dAdo to dATP by nucleoside kinase. It is interesting that nanomolar concentrations of ITu also completely protected chromaffin cells from 2'-dAdo lethality. Our study demonstrates for the first time that mammalian adrenal chromaffin cells undergo apoptotic cell death by a natural nucleoside and suggests that this model could be used to study apoptosis and cell function.     

A non-invasive technique for rapid extraction of DNA from fish scales

DNA markers are being increasingly used in studies related to population genetics and conservation biology of endangered species. DNA isolation for such studies requires a source of biological material that is easy to collect, non-bulky and reliable. Further, the sampling strategies based on non-invasive procedures are desirable, especially for the endangered fish species. In view of above, a rapid DNA extraction method from fish scales has been developed with the use of a modified lysis buffer that require about 2 hr duration. This methodology is non-invasive, less expensive and reproducible with high efficiency of DNA recovery. The DNA extracted by this technique, have been found suitable for performing restriction enzyme digestion and PCR amplification. Therefore, the present DNA extraction procedure can be used as an alternative technique in population genetic studies pertaining to endangered fish species. The technique was also found equally effective for DNA isolation from fresh, dried and ethanol preserved scales.