scholarly journals Inactivation during denaturation of ribonuclease A by guanidinium chloride is accompanied by unfolding at the active site

1995 ◽  
Vol 305 (2) ◽  
pp. 379-384 ◽  
Author(s):  
H J Yang ◽  
C L Tsou
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Ribonuclease A ◽  
Proteinase K ◽  
Peptide Fragments ◽  
Terminal Sequence ◽  
Guanidinium Chloride ◽  
Low Concentrations ◽  
Partial Unfolding ◽  
Absorption Measurements

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.

scholarly journals Conformational changes at the active site of creatine kinase at low concentrations of guanidinium chloride

1993 ◽  
Vol 291 (1) ◽  
pp. 103-107 ◽  
Author(s):  
H M Zhou ◽  
X H Zhang ◽  
Y Yin ◽  
C L Tsou
Keyword(s):  
Creatine Kinase ◽  
Fluorescent Probe ◽  
Conformational Change ◽  
Conformational Changes ◽  
Active Site ◽  
Emission Intensity ◽  
Enzyme Inactivation ◽  
Amino Groups ◽  
Guanidinium Chloride ◽  
Low Concentrations

It has been previously reported that, during denaturation of creatine kinase by guanidinium chloride (GdmCl) or urea [Tsou (1986), Trends Biochem. Sci. 11, 427-429], inactivation occurs before noticeable conformational change can be detected, and it is suggested that the conformation at the active site is more easily perturbed and hence more flexible than the molecule as a whole. In this study, the thiol and amino groups at or near the active site of creatine kinase are labelled with o-phthalaldehyde to form a fluorescent probe. Both the emission intensity and anisotropy decrease during denaturation indicating exposure of this probe and increased mobility of the active site. The above conformational changes take place together with enzyme inactivation at lower GdmCl concentrations than required to bring about intrinsic fluorescence changes of the enzyme. At the same GdmCl concentration, the rate of exposure of the probe is comparable with that of inactivation and is several orders of magnitude faster than that for the unfolding of the molecule as a whole.

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

1991 ◽  
Vol 277 (1) ◽  
pp. 207-211 ◽  
Author(s):  
Y Z Ma ◽  
C L Tsou
Keyword(s):  
Lactate Dehydrogenase ◽  
Active Site ◽  
Cross Linking ◽  
Native Conformation ◽  
Guanidinium Chloride ◽  
Original Activity ◽  
Limited Region ◽  
Low Concentrations ◽  
Two Stages ◽  
Inhibited Enzyme

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.

scholarly journals Conformational changes in gastric mucoproteins induced by caesium chloride and guanidinium chloride

1974 ◽  
Vol 141 (3) ◽  
pp. 641-646 ◽  
Author(s):  
David Snary ◽  
Adrian Allen ◽  
Roger H. Pain
Keyword(s):  
Molecular Weight ◽  
Conformational Changes ◽  
Sedimentation Coefficient ◽  
Water Structure ◽  
Water Soluble ◽  
Native Conformation ◽  
Guanidinium Chloride ◽  
Gastric Mucus ◽  
Caesium Chloride ◽  
Low Concentrations

1. Caesium chloride and guanidinium chloride were shown to cause conformational changes in the high-molecular-weight mucoprotein A of water-soluble gastric mucus with no change in molecular weight. 2. Increasing concentrations of CsCl decrease the viscosity of the mucoprotein bringing about a transition which is essentially complete in 0.1m-CsCl. The shear-dependence of viscosity of the mucoprotein is abolished by low concentrations of CsCl. The normally highly expanded molecule becomes contracted in CsCl to a molecule having the same symmetry but a smaller volume and decreased solvation, in keeping with an increased sedimentation coefficient (18.7S→33S). 3. This contracted form does not revert to the native conformation on removal of the CsCl. 4. A mechanism is discussed in terms of the effect of the Cs+and Cl−ions on water structure and the water–mucoprotein interaction. 5. Guanidinium chloride causes the CsCl-treated material to expand, in keeping with a decrease in s025,w (33S→26S). This is analogous to the known unfolding effect of guanidinium chloride on proteins and suggests that guanidinium chloride solubilizes groups involved in stabilizing the contracted structure. Removal of the guanidinium chloride results in a limited aggregation of four mucoprotein molecules. 6. These results show that caution must be exercised before interpreting the physical properties of mucoproteins which have been treated with CsCl and/or guanidinium chloride.

scholarly journals Activation of chicken liver dihydrofolate reductase by urea and guanidine hydrochloride is accompanied by conformational change at the active site

1996 ◽  
Vol 315 (1) ◽  
pp. 97-102 ◽  
Author(s):  
Ying-xin FAN ◽  
Ming JU ◽  
Jun-mei ZHOU ◽  
Chen-lu TSOU
Keyword(s):  
Catalytic Activity ◽  
Dihydrofolate Reductase ◽  
Conformational Changes ◽  
Active Site ◽  
Guanidine Hydrochloride ◽  
Chicken Liver ◽  
Peptide Fragments ◽  
Enhanced Fluorescence ◽  
Compact Structure ◽  
Mouse Leukaemia

It has been reported that the activation of dihydrofolate reductase (DHFR) from L1210 mouse leukaemia cells by KCl or thiol modifiers is accompanied by increased digestibility by proteinases [Duffy, Beckman, Peterson, Vitols and Huennekens (1987) J. Biol. Chem. 262, 7028–7033], suggesting a loosening up of the general compact structure of the enzyme. In the present study, the peptide fragments liberated from the chicken liver enzyme by digestion with trypsin in dilute solutions of urea or guanidine hydrochloride (GuHCl) have been separated by FPLC and sequenced. The sequences obtained are unique when compared with the known sequence of DHFR and thus allow the points of proteolytic cleavage identified for the urea- and GuHCl-activated enzyme to be at or near the active site. It was also indicated by the enhanced fluorescence of 2-p-toluidinylnaphthalene 6-sulphonate that conformational changes at the active site in dilute GuHCl parallel GuHCl activation. The above results indicate that the activation of DHFR in dilute denaturants is accompanied by a loosening up of its compact structure especially at or near the active site, suggesting that the flexibility at its active site is essential for the full expression of its catalytic activity.

The melanocyte stimulating activity of N-nitroso-2-chloroethyl-carbamoyl derivatives of α-melanotropin fragments

1988 ◽  
Vol 53 (11) ◽  
pp. 2574-2582 ◽  
Author(s):  
Hedvig Medzihradszky-Schweiger ◽  
Helga Süli-Vargha ◽  
József Bódi ◽  
Kálmán Medzihradszky
Keyword(s):  
Biological Activity ◽  
Active Site ◽  
Frog Skin ◽  
Terminal Sequence ◽  
Subsequent Reaction ◽  
Affinity Labels ◽  
Derivatives Of

A number of N-nitroso-2-chloroethyl-carbamoyl (Q(NO)) derivatives of α-melanotropin fragments have been synthesized and their effect on the frog skin melanocytes studied. Peptides substituted in this way possess the biological activity of the parent compounds, indicating that they preserved their receptor recognizing ability. These compounds can therefore serve as affinity labels. Some of these derivatives, related to the C-terminal sequence of α-melanotropin show prolonged darkening reaction, which does not influence the subsequent reaction of melanocytes with α-melanotropin. The Q(NO)-derivative of a fragment derived from the classical active site of the hormone shows, however, inhibition of the effect of α-melanotropin. It can be concluded that the latter peptide acts through the melanotropin receptor, while others, related to the C-terminal sequence of the hormone through another mechanism.

scholarly journals E3 Ubiquitin Ligase SPL2 Is a Lanthanide-Binding Protein

2021 ◽  
Vol 22 (11) ◽  
pp. 5712
Author(s):  
Michał Tracz ◽  
Ireneusz Górniak ◽  
Andrzej Szczepaniak ◽  
Wojciech Białek
Keyword(s):  
Ubiquitin Ligase ◽  
Conformational Changes ◽  
Protein Interactions ◽  
E3 Ubiquitin Ligase ◽  
Lanthanide Ions ◽  
E3 Ligases ◽  
Fluorescence Measurements ◽  
Partial Unfolding

The SPL2 protein is an E3 ubiquitin ligase of unknown function. It is one of only three types of E3 ligases found in the outer membrane of plant chloroplasts. In this study, we show that the cytosolic fragment of SPL2 binds lanthanide ions, as evidenced by fluorescence measurements and circular dichroism spectroscopy. We also report that SPL2 undergoes conformational changes upon binding of both Ca2+ and La3+, as evidenced by its partial unfolding. However, these structural rearrangements do not interfere with SPL2 enzymatic activity, as the protein retains its ability to auto-ubiquitinate in vitro. The possible applications of lanthanide-based probes to identify protein interactions in vivo are also discussed. Taken together, the results of this study reveal that the SPL2 protein contains a lanthanide-binding site, showing for the first time that at least some E3 ubiquitin ligases are also capable of binding lanthanide ions.

scholarly journals Active site lysine backbone undergoes conformational changes in the bacteriorhodopsin photocycle.

1994 ◽  
Vol 269 (10) ◽  
pp. 7387-7389
Author(s):  
H. Takei ◽  
Y. Gat ◽  
Z. Rothman ◽  
A. Lewis ◽  
M. Sheves
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Bacteriorhodopsin Photocycle

A Deuterium-Hydrogen Exchange Study of Inhibitor-Induced Conformational Changes in Ribonuclease A

1971 ◽  
Vol 21 (3) ◽  
pp. 393-399 ◽  
Author(s):  
Genevieve Nonnenmacher ◽  
Eliane Viala ◽  
Jean Marie Thiery ◽  
Paul Calvet
Keyword(s):  
Conformational Changes ◽  
Hydrogen Exchange ◽  
Ribonuclease A ◽  
Exchange Study
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