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The denaturation of rabbit muscle phosphorylase b by guanidinium chloride.

ABSTRACT: The denaturation of phosphorylase b by guanidinium chloride (GdnHCl) was studied. The enzyme is unusually sensitive to the denaturing agent, being more than 50% inactivated after incubation for 15 min in 0.1 M-GdnHCl. Full activity can be regained on dilution of the GdnHCl to 0.01 M, provided that the initial concentration of GdnHCl is less than 0.5 M. Studies of protein fluorescence, thiol-group reactivity, circular dichroism and absorption spectroscopy indicate that phosphorylase b undergoes slow structural changes in the range of GdnHCl concentrations from 0.5 to 0.8 M. The enzyme retains considerable folded structure even after 15 min incubation in 1 M-GdnHCl, but is rapidly and completely unfolded in 3 M-GdnHCl.

SUBMITTER: Price NC

PROVIDER: S-EPMC1152173 | biostudies-other | 1983 Sep

REPOSITORIES: biostudies-other

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Kinetic evidence for a two-stage mechanism of protein denaturation by guanidinium chloride.

Project description:Dry molten globular (DMG) intermediates, an expanded form of the native protein with a dry core, have been observed during denaturant-induced unfolding of many proteins. These observations are counterintuitive because traditional models of chemical denaturation rely on changes in solvent-accessible surface area, and there is no notable change in solvent-accessible surface area during the formation of the DMG. Here we show, using multisite fluorescence resonance energy transfer, far-UV CD, and kinetic thiol-labeling experiments, that the guanidinium chloride (GdmCl)-induced unfolding of RNase H also begins with the formation of the DMG. Population of the DMG occurs within the 5-ms dead time of our measurements. We observe that the size and/or population of the DMG is linearly dependent on [GdmCl], although not as strongly as the second and major step of unfolding, which is accompanied by core solvation and global unfolding. This rapid GdmCl-dependent population of the DMG indicates that GdmCl can interact with the protein before disrupting the hydrophobic core. These results imply that the effect of chemical denaturants cannot be interpreted solely as a disruption of the hydrophobic effect and strongly support recent computational studies, which hypothesize that chemical denaturants first interact directly with the protein surface before completely unfolding the protein in the second step (direct interaction mechanism).

| S-EPMC3977270 | biostudies-literature

Protein Denaturation with Guanidinium: A 2D-IR Study.

Project description:Guanidinium (Gdm+) is a widely used denaturant, but it is still largely unknown how it operates at the molecular level. In particular, the effect of guanidinium on the different types of secondary structure motifs of proteins is at present not clear. Here, we use two-dimensional infrared spectroscopy (2D-IR) to investigate changes in the secondary structure of two proteins with mainly α-helical or β-sheet content upon addition of Gdm-13C15N3·Cl. We find that upon denaturation, the β-sheet protein shows a complete loss of β-sheet structure, whereas the α-helical protein maintains most of its secondary structure. These results suggest that Gdm+ disrupts β-sheets much more efficiently than α-helices, possibly because in the former, hydrophobic interactions are more important and the number of dangling hydrogen bonds is larger.

| S-EPMC3805561 | biostudies-literature

Comparison of the activity and conformation changes of lactate dehydrogenase H4 during denaturation by guanidinium chloride.

Project description:The inactivation and unfolding of lactate dehydrogenase (LDH) during denaturation by guanidinium chloride (GuHCl) under diverse conditions have been compared. Unfolding of the native conformation, as monitored by fluorescence and c.d. measurements, occurs in two stages with increasing GuHCl concentrations, and the inactivation approximately coincides with, but slightly precedes, the first stage of unfolding. The enzyme is inhibited to about 60-70% of its original activity by cross-linking with glutaraldehyde or in the presence of 1 M-(NH4)2SO4, with its conformation stabilized as shown by the requirement for higher GuHCl concentrations to bring about both inactivation and unfolding. Low concentrations of GuHCl (0.2-0.4 M) activate the cross-linked and the (NH4)2SO4-inhibited enzyme back to the level of the native enzyme. For the enzyme stabilized by (NH4)2SO4 or by cross-linking with glutaraldehyde, inactivation occurs at a markedly lower GuHCl concentration than that required to bring about its first stage of unfolding. It is concluded that the active site of LDH is situated in a limited region relatively fragile in conformation as compared with the molecule as a whole. The GuHCl activation of LDH stabilized in (NH4)2SO4 or by cross-linking with glutaraldehyde suggests that this fragility and consequently flexibility of the active site is required for its catalytic activity.

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Project description:Inactivation of pancreatic RNAase A occurs in guanidinium chloride (GdmCl) at low concentrations before the unfolding of the molecule as a whole can be detected [Liu and Tsou (1987) Biochim. Biophys. Acta 916, 455-464]. We have now shown that the rate of digestion of the RNAase molecule by either trypsin or proteinase K increases significantly at low concentrations of GdmCl where the enzyme is largely inactivated, but fluorescence and absorption measurements reveal no conformational changes. N-Terminal sequence analysis of the peptide fragments generated shows that proteolysis occurs primarily at or near the active site. The decrease in activity of RNAase at low concentrations of GdmCl is therefore due to partial unfolding of the molecule, particularly at the active site and not to an inhibition by the denaturant.

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2-(1,3-Benzoxazol-2-yl)guanidinium chloride.

Project description:The non-H atoms of the cation of the title salt, C(8)H(9)N(4)O(+)·Cl(-), are approximately co-planar (r.m.s. deviation = 0.024?Å) with one amino group forming an intra-molecular hydrogen bond to the tertiary N atom of the benzoxazole fused-ring system. The cations and anions are linked by cyclic R(2) (1)(6) N-H?Cl hydrogen-bonding associations, generating linear chains running along the a-axis direction.

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N-Benzoyl-N',N''-diphenyl-guanidinium chloride.

Project description:In the title compound, C(20)H(18)N(3)O(+)·Cl(-), the orientation of the aromatic rings around the planar CN(3) (+) unit produces steric hindrance. As a consequence of this particular orientation of the guanidinium cation, hydrogen bonding is restricted to N-H⋯Cl and intra-molecular N-H⋯O hydrogen bonds within the discrete unit. The guanidinium and carbonyl groups are coplanar as a result of the six-membered ring formed by the N-H⋯O intra-molecular hydrogen bond. The dihedral angles between the guanidinium plane and the two phenyl rings are 62.31 (8) and 64.24 (8)°.

| S-EPMC2915374 | biostudies-literature

Guanidinium chloride-18-crown-6 (2/1).

Project description:In the crystal of the title compound, 2CH(6)N(3) (+)·2Cl(-)·C(12)H(24)O(6), the 18-crown-6 mol-ecule is located across an inversion center. The guanidinium cation links to the 18-crown-6 mol-ecule and chloride anion via N-H⋯O and N-H⋯Cl hydrogen bonds.

| S-EPMC3344600 | biostudies-literature

Changes in water structure induced by the guanidinium cation and implications for protein denaturation.

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| S-EPMC2646201 | biostudies-literature

Inactivation precedes changes in allosteric properties and conformation of D-glyceraldehyde-3-phosphate dehydrogenase and fructose-1,6-bisphosphatase during denaturation by guanidinium chloride.

Project description:It has been shown that inactivation of several enzymes precedes overall conformational changes of the enzyme molecules as a whole during denaturation [Tsou (1993) Science, 262, 380-381]. However, the relation between inactivation, loss of allosteric properties of oligomeric enzymes and unfolding of the enzyme molecule during denaturation remain little explored. These have now been compared for D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and fructose-1,6-bisphosphatase (FruP2ase) during denaturation by guanidinium chloride (GdmCl). GAPDH is completely inactivated at 0.3 M GdmCl but at this GdmCl concentration it still binds NAD+ with negative co-operativity. At 0.4 M GdmCl, inactivation of FruP2ase reaches completion whereas its allosteric properties, including the heterotropic effect of AMP inhibition and K+ activation with positive co-operativity, are only partially affected. Much higher GdmCl concentrations are required to bring about unfolding of the overall structures of both enzymes.

| S-EPMC1137582 | biostudies-other

Liquid-vapor interfacial properties of aqueous solutions of guanidinium and methyl guanidinium chloride: influence of molecular orientation on interface fluctuations.

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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