Unfolding and refolding of phospholipase C from Bacillus cereus in solutions of guanidinium chloride.
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ABSTRACT:
SUBMITTER: Little C
PROVIDER: S-EPMC1186658 | biostudies-other | 1979 Jun
REPOSITORIES: biostudies-other
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Project description:Delta-crystallin is the major soluble protein in avian eye lenses with a structural role in light scattering. Dissociation and unfolding of the tetrameric protein in guanidinium chloride (GdmCl) can be sensitively monitored by the intrinsic tryptophan fluorescence. In this study refolding of GdmCl-denatured delta-crystallin was investigated. A marked hysteresis was observed while refolding by dilution of the 5 M GdmCl-denatured delta-crystallin. The secondary structure of the refolded protein was largely restored. However, monitoring intrinsic fluorescence of single tryptophan mutants indicated that the microenvironment of domain 1 (W74) was not restored. The region containing W169, which is close to the dimer interface, remained exposed following refolding. During refolding of the wild-type protein, dimeric, tetrameric, and aggregate forms were identified. The ratio of tetramer to dimer increased with time, as judged by gel-filtration chromatography and nondenaturing gel electrophoresis. However the observed levels of tetramer did not return to the same levels as observed before GdmCl treatment. The proportion of tetramer was significantly decreased in the N-25 deletion mutant and it did not increase with time. These results suggest that there is a kinetic barrier for assembly of dimers into tetramers. The consequence of this is that dimers refold to form aggregates. Aggregation seems to follow a nucleation mechanism with an apparent reaction order of 4.7+/-0.2, suggesting four or five monomers constitute the core structure of nucleus, which propagate to form high molecular weight aggregates. Addition of alpha-crystallin during refolding prevents aggregation. Thioflavin T and Congo red assays indicated a regular structure for the protein aggregates, which appear as hollow tubules packed into helical bundles. Aggregate formation was protein concentration dependent that progressed via two stages with rate constants of 0.0039+/-0.0006 and 0.00043+/-0.00003 s(-1), respectively. We propose that the N-terminal segment of delta-crystallin plays a critical role in proper double dimer assembly and also in the assembly of nucleus to aggregate formation.
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Project description:The effect of guanidinium chloride (GdnHCl) on the pyruvate dehydrogenase complex (PDC) from bovine heart and its constituent enzymes has been studied. The overall activity of the complex is lost reversibly at low levels of GdnHCl (0.2 M) which cause 40-50% inactivation but no loss of overall secondary or tertiary structures of the individual enzymes; the inactivation of the complex is shown to be caused by dissociation of the E1 and E3 components from the E2/X core assembly. This provides an improved procedure for controlled dissociation of the complex and efficient recovery of its component enzymes in their native states. Higher concentrations of GdnHCl (up to 4 M) lead to the unfolding and irreversible inactivation of the separate enzymes of the complex with the E2/X core proving the most resistant to GdnHCl-induced unfolding. Neither the 60-meric E2/X core assembly nor the dimeric E3 component are dissociated into monomers in the presence of 6 M GdnHCl; the latter enzyme forms higher-M(r) aggregates under these conditions.
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Project description:The denatured states of proteins have always attracted our attention due to the fact that the denatured state is the only experimentally achievable state of a protein, which can be taken as initial reference state for considering the in vitro folding and defining the native protein stability. It is known that heat and guanidinium chloride (GdmCl) give structurally different states of RNase-A, lysozyme, ?-chymotrypsinogen A and ?-lactalbumin. On the contrary, differential scanning calorimetric (DSC) and isothermal titration calorimetric measurements, reported in the literature, led to the conclusion that heat denatured and GdmCl denatured states are thermodynamically and structurally identical. In order to resolve this controversy, we have measured changes in the far-UV CD (circular dichroism) of these heat-denatured proteins on the addition of different concentrations of GdmCl. The observed sigmoidal curve of each protein was analyzed for Gibbs free energy change in the absence of the denaturant (?G0X?D) associated with the process heat denatured (X) state ? GdmCl denatured (D) state. To confirm that this thermodynamic property represents the property of the protein alone and is not a manifestation of salvation effect, we measured urea-induced denaturation curves of these heat denatured proteins under the same experimental condition in which GdmCl-induced denaturation was carried out. In this paper we report that (a) heat denatured proteins contain secondary structure, and GdmCl (or urea) induces a cooperative transition between X and D states, (b) for each protein at a given pH and temperature, thermodynamic cycle connects quantities, ?G0N?X (native (N) state ? X state), ?G0X?D and ?G0N?D (N state ? D state), and
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Project description:Cosolvents play an important role in regulating the stability and function of proteins present in the cell. We studied the role of cosolvents, urea and guanidinium chloride (GdmCl), which act as protein denaturants, in the catalytic activity and structural stability of the protein lysozyme using activity measurements, spectroscopy, and molecular dynamics simulations. We find that the activity of lysozyme increases on the addition of urea, whereas it decreases sharply on the addition of GdmCl. At low GdmCl concentrations ([GdmCl] < 4 M), the activity of lysozyme decreases, even though there is no significant perturbation in the structure of the lysozyme folded state. We find that this is due to the strong interaction of the Gdm+ ion with the residues Asp52 and Glu35, which are present in the lysozyme catalytic site. In contrast, urea interacts with Trp63 present in the loop region present near the active site of lysozyme, inducing minor conformational changes in lysozyme, which can increase the activity of lysozyme. At higher denaturant concentrations, experiments show that GdmCl completely denatures the protein, whereas the folded state is stable in the presence of urea. We further show that GdmCl denatures lysozyme with the disulfide bonds intact in the protein, whereas urea denatures the protein only when the disulfide bonds are broken using reducing agents.
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Project description:Chemical denaturant sensitivity of the dimeric main protease from severe acute respiratory syndrome (SARS) coronavirus to guanidinium chloride was examined in terms of fluorescence spectroscopy, circular dichroism, analytical ultracentrifuge, and enzyme activity change. The dimeric enzyme dissociated at guanidinium chloride concentration of <0.4 M, at which the enzymatic activity loss showed close correlation with the subunit dissociation. Further increase in guanidinium chloride induced a reversible biphasic unfolding of the enzyme. The unfolding of the C-terminal domain-truncated enzyme, on the other hand, followed a monophasic unfolding curve. Different mutants of the full-length protease (W31 and W207/W218), with tryptophanyl residue(s) mutated to phenylalanine at the C-terminal or N-terminal domain, respectively, were constructed. Unfolding curves of these mutants were monophasic but corresponded to the first and second phases of the protease, respectively. The unfolding intermediate of the protease thus represented a folded C-terminal domain but an unfolded N-terminal domain, which is enzymatically inactive due to loss of regulatory properties. The various enzyme forms were characterized in terms of hydrophobicity and size-and-shape distributions. We provide direct evidence for the functional role of C-terminal domain in stabilization of the catalytic N-terminal domain of SARS coronavirus main protease.
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Project description:The guanidinium cation (C(NH2)3(+)) is a highly stable cation in aqueous solution due to its efficient solvation by water molecules and resonance stabilization of the charge. Its salts increase the solubility of nonpolar molecules ("salting-in") and decrease the ordering of water. It is one of the strongest denaturants used in biophysical studies of protein folding. We investigate the behavior of guanidinium and its derivative, methyl guanidinium (an amino acid analogue) at the air-water surface, using atomistic molecular dynamics (MD) simulations and calculation of potentials of mean force. Methyl guanidinium cation is less excluded from the air-water surface than guanidinium cation, but both cations show orientational dependence of surface affinity. Parallel orientations of the guanidinium ring (relative to the Gibbs dividing surface) show pronounced free energy minima in the interfacial region, while ring orientations perpendicular to the GDS exhibit no discernible surface stability. Calculations of surface fluctuations demonstrate that, near the air-water surface, the parallel-oriented cations generate significantly greater interfacial fluctuations compared to other orientations, which induces more long-ranged perturbations and solvent density redistribution. Our results suggest a strong correlation with induced interfacial fluctuations and ion surface stability. These results have implications for interpreting molecular-level, mechanistic action of this osmolyte's interaction with hydrophobic interfaces as they impact protein denaturation (solubilization).
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Project description:The macromolecular properties of cervical-mucus glycoproteins (mucins) were studied as a function of the concentration of guanidinium chloride with conventional light-scattering, photon-correlation spectroscopy and sedimentation-velocity centrifugation. No evidence for an association of the mucins in 0.2M-NaCl as compared with 6M-guanidinium chloride was found at mucin concentrations below approx. 0.5 mg/ml. However, an increase in the frictional coefficient and in the radius of gyration occurred with increasing concentrations of guanidinium chloride, in particular between 4 M and 6 M, suggesting an expansion of the individual macromolecule. The change in the particle-scattering function is consistent with a transition from a 'stiff' random coil in 0.2 M-NaCl into a more flexible one in 6 M-guanidinium chloride. We suggest that the mucins contain regions of 'ordered' structure which can undergo a reversible 'unfolding' analogous to the behaviour of a conventional globular protein exposed to a denaturing solvent. Such regions might carry sites for specific interactions between mucins and/or be decisive for their conformation and macromolecular properties in physiological solvents.
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Project description:The rate of phospholipid hydrolysis in erythrocyte ghosts by Bacillus cereus phospholipase C was markedly decreased by the presence of NaCl at concentrations between 25 and 200 mM. The inhibition seemed to be due to Cl- and was unaffected by the type of cation present. The larger univalent anions such as HCO3-, Br-, Cl-, NO3-, CNO- and I- seemed most effective, whereas the bivalent anion SO42- was relatively ineffective at 0.1 M, as were acetate and formate. Tris buffers at 0.1 M caused marked inhibition. With bovine brain myelin, phospholipid hydrolysis by phospholipase C was also much more strongly inhibited by I- and Cl- than by SO42- or acetate. NaCl inhibited the hydrolysis by the enzyme of the soluble substrate dihexanoylglycerophosphocholine, thereby suggesting that the inhibiton did not arise simply from substrate effects.
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Project description:The inactivation of phospholipase C from Bacillus cereus at pH6 by diethyl pyrocarbonate parallelled the N-ethoxyformylation of a single histidine residue in the enzyme. The inactivation arose from a decrease in the maximum velocity of the enzymic reaction with no effect on the Km value. The inactivation did not apparently alter the ability of the enzyme to bind to a substrate-based affinity gel. The native enzyme contained only one reactive histidine residue. Removal of the two zinc atoms from the enzyme increased the number of reactive histidine residues to five, whereas in the totally denatured enzyme nearly eight such residues were available for reaction with diethyl pyrocarbonate. The enzyme thus appears to contain one histidine residue that is essential for catalytic activity and four that may be involved in co-ordinating the zinc atoms in the structure.
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Project description:1. Phospholipase C was inactivated by exposure to the three amino-group reagents, ethyl acetamidate, 2,4,6-trinitrobenzensulphonic acid and pyridoxal 5'-phosphate plus reduction. 2. Inactivation by pyridoxal 5'-phosphate showed the characteristics of Schiff's base formation with the enzyme. The pyridoxal 5'-phosphate-treated enzyme after reduction had an absorbance maximum at 325 mm and 6-N-pyridoxyl-lysine was the only fluorescent component after acid hydrolysis. 3. For complete inactivation, 2 mol of pyridoxal 5'-phosphate or 7 mol of 2,4,6-trinitrophenyl were incorporated/mol of enzyme. 4. The two apparently essential lysine residues were much more reactive to pyridoxal 5'-phosphate than the other 19 lysine residues in the enzyme. 5. Binding of phospholipase C to a substrate-based affinity gel caused marked protection against inactivation by pyridoxal 5'-phosphate. For complete inactivation of the gel-bound enzyme, 5 mol of pyridoxal 5'-phosphate were incorporated/mol of enzyme and there was no evidence of two especially reactive lysine residues. 6. On application of pyridoxal 5'-phosphate-treated enzyme (remaining activity 30% of original) to a column of the affinity gel, some material bound and some did not. The latter contained very little enzyme activity and was heavily incorporated with reagent (9.06 mol/mol of enzyme). The former had a specific activity of 34% of that of the control and contained 1.29 mol of reagent/mol of enzyme. 7. Thus phospholipase C appears to contain two lysine residues that are essential for enzyme activity, but probably not for substrate binding.
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