The equilibrium and kinetic unfolding properties of homodimeric class alpha glutathione transferase (hGST A1-1) were characterized. Urea-induced equilibrium unfolding data were consistent with a folded dimer/unfolded monomer transition. Unfolding kinetics were investigated, using stopped-flow fluorescence, as a function of denaturant concentration (3.5-8.9 M urea) and temperature (10-40 degrees C). The unfolding pathway, monitored by tryptophan fluorescence, was biphasic with a fast unfolding event (millisecond time range with enhanced fluorescence properties) and a slow unfolding event (seconds to minutes time range with quenched fluorescence properties). Both events occurred simultaneously from 3.5 M urea. Each phase displayed single-exponential behavior, consistent with two unimolecular reactions. Urea-dependence studies and thermodynamic activation parameters (transition-state theory) suggest that the transition state for each phase is well-structured and is closely related to native protein in terms of solvent exposure. The apparent activation Gibbs free energy change in the absence of denaturant, DeltaG (H2O), indicates that the slow unfolding event represents the transition state for the overall unfolding pathway. The rate and urea independence of each phase on the initial condition exclude the possibility of a preexisting equilibrium between various native forms in the pretransition baseline. The unfolding pathways monitored by energy transfer to or direct excitation of AEDANS covalently linked to Cys111 in hGST A1-1 were monophasic with urea and temperature properties similar to those observed for the slow unfolding event (described above). A sequential unfolding kinetic mechanism involving the partial dissociation of the two structurally distinct domains per subunit followed by complete domain and subunit unfolding is proposed.
The conformational stabilities of two homodimeric class mu glutathione transferases (GSTM1-1 and GSTM2-2) were studied by urea- and guanidinium chloride-induced denaturation. Unfolding is reversible and structural changes were followed with far-ultraviolet circular dichroism, tryptophan fluorescence, enzyme activity, chemical cross-linking, and size-exclusion chromatography. Disruption of secondary structure occurs as a monophasic transition and is independent of protein concentration. Changes in tertiary structure occur as two transitions; the first is protein concentration dependent, while the second is weakly dependent (GSTM1-1) or independent (GSTM2-2). The second transition corresponds with the secondary structure transition. Loss in catalytic activity occurs as two transitions for GSTM1-1 and as one transition for GSTM2-2. These transitions are dependent upon protein concentration. The first deactivation transition coincides with the first tertiary structure transition. Dimer dissociation occurs prior to disruption of secondary structure. The data suggest that the equilibrium unfolding/refolding of the class mu glutathione transferases M1-1 and M2-2 proceed via a three-state process: N(2) <--> 2I <--> 2U. Although GSTM1-1 and GSTM2-2 are homologous (78% identity/94% homology), their N(2) tertiary structures are not identical. Dissociation of the GSTM1-1 dimer to structured monomers (I) occurs at lower denaturant concentrations than for GSTM2-2. The monomeric intermediate for GSTM1-1 is, however, more stable than the intermediate for GSTM2-2. The intermediates are catalytically inactive and display nativelike secondary structure. Guanidinium chloride-induced denaturation yields monomeric intermediates, which have a more loosely packed tertiary structure displaying enhanced solvent exposure of its tryptophans and enhanced ANS binding. The three-state model for the class mu enzymes is in contrast to the equilibrium two-state models previously proposed for representatives of classes alpha/pi/Sj26 GSTs. Class mu subunits appear to be intrinsically more stable than those of the other GST classes.
Many proteins fold through intermediates that are populated in the submillisecond time regime. To monitor directly the formation of these kinetic intermediates, we have developed a simple, robust, easy to assemble continuous flow mixer for studying folding reactions in the 35–1000μs time regime. The mixer is constructed by laser-machining 75-μm channels in a 127-μm-thick polyimide or polyetheretherketone polymer wafer. Mixing times of ∼25to∼50μs can be achieved for a 1∕10 dilution reaction of 8M urea with flow rates of 10–20mL∕min. CCD-based steady-state and time-correlated single-photon-counting-based fluorescence detection strategies are described. Preliminary results on the early events in the refolding of cytochrome c are presented.
Here we report the discovery, by high-throughput screening, of three novel (2-amino-5-keto)thiazole compounds that act as selective potentiators of nicotinic acetylcholine receptors. Compound selectivity was assessed at seven human nicotinic acetylcholine receptors (alpha1beta1gammadelta, alpha2beta4, alpha3beta2, alpha3beta4, alpha4beta2, alpha4beta4, and alpha7) expressed in mammalian cells or Xenopus oocytes. At alpha2beta4, alpha4beta2, alpha4beta4, and alpha7, but not alpha1beta1gammadelta, alpha3beta2, or alpha3beta4, submaximal responses to nicotinic agonists were potentiated in a concentration-dependent manner by all compounds. At similar concentrations, no potentiation of 5-hydroxytryptamine, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, GABA(A), and N-methyl-d-aspartate receptors or voltage-gated Na(+) and Ca(2+) channels was observed. Furthermore, these compounds did not inhibit acetylcholine esterase. Further profiling revealed that these compounds enhanced the potency and maximal efficacy of a range of nicotinic agonists at alpha4beta2 nicotinic acetylcholine receptors, a profile typical of allosteric potentiators. At concentrations required for potentiation, the compounds did not displace [(3)H]epibatidine from the agonist-binding site, and potentiation was observed at all agonist concentrations, suggesting a noncompetitive mechanism of action. Blockade of common second messenger systems did not affect potentiation. At concentrations higher then required for potentiation the compounds also displayed intrinsic agonist activity, which was blocked by competitive and noncompetitive nicotinic acetylcholine receptor (nAChR) antagonists. These novel selective nicotinic receptor potentiators should help in clarifying the potential therapeutic utility of selective nAChR modulation for the treatment of central nervous system disorders.
Helix 9 at the C-terminus of class alpha glutathione transferase (GST) polypeptides is a unique structural feature in the GST superfamily. It plays an important structural role in the catalytic cycle. Its contribution toward protein stability/folding as well as the binding of nonsubstrate ligands was investigated by protein engineering, conformational stability, enzyme activity, and ligand-binding methods. The helix9 sequence displays an unfavorable propensity toward helix formation, but tertiary interactions between the amphipathic helix and the GST seem to contribute sufficient stability to populate the helix on the surface of the protein. The helix's stability is enhanced further by the binding of ligands at the active site. The order of ligand-induced stabilization increases from H-site occupation, to G-site occupation, to the simultaneous occupation of H- and G-sites. Ligand-induced stabilization of helix9 reduces solvent accessible hydrophobic surface by facilitating firmer packing at the hydrophobic interface between helix and GST. This stabilized form exhibits enhanced affinity for the binding of nonsubstrate ligands to ligandin sites (i.e., noncatalytic binding sites). Although helix9 contributes very little toward the global stability of hGSTA1-1, its conformational dynamics have significant implications for the protein's equilibrium unfolding/refolding pathway and unfolding kinetics. Considering the high concentration of reduced glutathione in human cells (about 10 mM), the physiological form of hGSTA1-1 is most likely the thiol-complexed protein with a stabilized helix9. The C-terminus region (including helix9) of the class alpha polypeptide appears not to have been optimized for stability but rather for catalytic and ligandin function.
A hydrophobic lock-and-key intersubunit motif involving a phenylalanine is a major structural feature conserved at the dimer interface of classes alpha, mu and pi glutathione transferases. In order to determine the contribution of this subunit interaction towards the function and stability of human class alpha GSTA1-1, the interaction was truncated by replacing the phenylalanine`key' Phe-51 with serine. The F51S mutant protein is dimeric with a native-like core structure indicating that Phe-51 is not essential for dimerization. The mutation impacts on catalytic and ligandin function suggesting that tertiary structural changes have occurred at/near the active and non-substrate ligand-binding sites. The active site appears to be disrupted mainly at the glutathione-binding region that is adjacent to the lock-and-key intersubunit motif. The F51S mutant displays enhanced exposure of hydrophobic surface and ligandin function. The lock-and-key motif stabilizes the quaternary structure of hGSTA1-1 at the dimer interface and the protein concentration dependence of stability indicates that the dissociation and unfolding processes of the mutant protein remain closely coupled.z 2000 Federation of European Biochemical Societies.
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