Kinetics of Actin Unfolding Induced by Guanidine Hydrochloride

Turoverov, Konstantin K.; Verkhusha, Vladislav V.; Shavlovsky, M. M.; Biktashev, Alexander G.; Povarova, Olga I.; Kuznetsova, Irina M.

doi:10.1021/bi015548c

Cited by 38 publications

(69 citation statements)

References 43 publications

(70 reference statements)

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“…Protein solutions containing a desired denaturant concentration, with or without crowding agent at the desired concentration, were incubated at 23 °C for 24 h. It is worth noting that thus-measured characteristics of the protein are quasi-equilibrium parameters [15]. According to the control experiments, the dead-time in these manual mixing-based unfolding-refolding experiments was about 4 s [53,54]. …”

Section: Methodsmentioning

confidence: 99%

Peculiarities of the Super-Folder GFP Folding in a Crowded Milieu

Stepanenko

Kuznetsova

Uversky

et al. 2016

IJMS

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The natural cellular milieu is crowded by large quantities of various biological macromolecules. This complex environment is characterized by a limited amount of unoccupied space, limited amounts of free water, and changed solvent properties. Obviously, such a tightly packed cellular environment is poorly mimicked by traditional physiological conditions, where low concentrations of a protein of interest are analyzed in slightly salted aqueous solutions. An alternative is given by the use of a model crowded milieu, where a protein of interest is immersed in a solution containing high concentrations of various polymers that serve as model crowding agents. An expected outcome of the presence of such macromolecular crowding agents is their ability to increase conformational stability of a globular protein due to the excluded volume effects. In line with this hypothesis, the behavior of a query protein should be affected by the hydrodynamic size and concentration of an inert crowder (i.e., an agent that does not interact with the protein), whereas the chemical nature of a macromolecular crowder should not play a role in its ability to modulate conformational properties. In this study, the effects of different crowding agents (polyethylene glycols (PEGs) of various molecular masses (PEG-600, PEG-8000, and PEG-12000), Dextran-70, and Ficoll-70) on the spectral properties and unfolding–refolding processes of the super-folder green fluorescent protein (sfGFP) were investigated. sfGFP is differently affected by different crowders, suggesting that, in addition to the expected excluded volume effects, there are some changes in the solvent properties.

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Section: Methodsmentioning

confidence: 99%

Peculiarities of the Super-Folder GFP Folding in a Crowded Milieu

Stepanenko

Kuznetsova

Uversky

et al. 2016

IJMS

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“…On the other hand, Parameter A, being the ratio of two points of the spectra, indicates if there are spectral variations independently of the position of the peak. For example, if two different conformational states are responsible for two different spectra, this could not be noted if only l max of emission is taken into consideration (Turoverov et al, 2002). Therefore, since both Dl max and Parameter A vary as a function of MeOH concentration, it can be deduced that the organic solvent causes conformational changes in both CHMO and PAMO.…”

Section: Fluorescence Measurementsmentioning

confidence: 99%

Effects of water miscible organic solvents on the activity and conformation of the baeyer–villiger monooxygenases from Thermobifida fusca and Acinetobacter calcoaceticus: A comparative study

Secundo

Fiala

Fraaije

et al. 2010

Biotech & Bioengineering

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A broader exploitation of enzymes in organic synthesis can be achieved by increasing their tolerance toward organic solvents. In this study, the stability and activity of Baeyer-Villiger monooxygenases from Thermobifida fusca (PAMO) and Acinetobacter sp. (CHMO) in the presence of water miscible organic solvents were compared. PAMO was more stable than CHMO. The concentration of solvent (v/v) at which it halved its activity (C(50) ) was 4- to 16-fold higher than that observed for CHMO. For PAMO, the C(50) varied from 16% to 55% of solvent and followed the destabilizing order methanol < ethanol < 1,4-dioxane < acetonitrile < trifluoroethanol. In the case of CHMO, the maximal C(50) was 7% with methanol and even lower with the other solvents. Therefore, methanol was the most tolerated solvent. In the case of PAMO, methanol induced a significant increase of enzyme activity (up to fivefold), which was optimal at 20% (v/v) solvent. Only minor spectral variations were observed with PAMO in 20% methanol, suggesting that the increase of activity observed in this condition is not due to marked conformational changes. Fluorescence and circular dichroism analyses showed that the lower stability of CHMO toward organic solvent correlates with a more pronounced destructive effect on its secondary and tertiary structure. A possible rationale for the higher stability of PAMO could be inferred from inspection of the PAMO and CHMO (two enzymes of similar size) structure, which revealed a higher (up to twofold) number of ionic bridges in PAMO with respect to CHMO.

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“…For the first time, protein aggregation in the solution of low concentration of GdnHCl we have revealed for actin. In this case the effect is especially pronounced because in aggregation are involved large supramolecular complexes of inactivated actin [11], [12] and it is accompanied not only by the increase in ANS fluorescence intensity, but by the increase in light scattering also.…”

Section: Introductionmentioning

confidence: 98%

Differences in the Pathways of Proteins Unfolding Induced by Urea and Guanidine Hydrochloride: Molten Globule State and Aggregates

2010

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It was shown that at low concentrations guanidine hydrochloride (GdnHCl) can cause aggregation of proteins in partially folded state and that fluorescent dye 1-anilinonaphthalene-8-sulfonic acid (ANS) binds with these aggregates rather than with hydrophobic clusters on the surface of protein in molten globule state. That is why the increase in ANS fluorescence intensity is often recorded in the pathway of protein denaturation by GdnHCl, but not by urea. So what was previously believed to be the molten globule state in the pathway of protein denaturation by GdnHCl, in reality, for some proteins represents the aggregates of partially folded molecules.

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Kinetics of Actin Unfolding Induced by Guanidine Hydrochloride

Cited by 38 publications

References 43 publications

Peculiarities of the Super-Folder GFP Folding in a Crowded Milieu

Peculiarities of the Super-Folder GFP Folding in a Crowded Milieu

Effects of water miscible organic solvents on the activity and conformation of the baeyer–villiger monooxygenases from Thermobifida fusca and Acinetobacter calcoaceticus: A comparative study

Differences in the Pathways of Proteins Unfolding Induced by Urea and Guanidine Hydrochloride: Molten Globule State and Aggregates

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