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Structural characteristic of the initial unfolded state on refolding determines catalytic efficiency of the folded protein in presence of osmolytes.

Warepam M, Sharma GS, Dar TA, Khan MK, Singh LR - PLoS ONE (2014)

Bottom Line: The interaction of osmolyte with the native state does not significantly contribute to the osmolyte-induced protein folding.We have therefore investigated if different denatured states of a protein (generated by different denaturing agents) interact differently with the osmolytes to induce protein folding.These conclusions have been reached from the systematic measurements of enzymatic kinetic parameters (Km and kcat), thermodynamic stability (Tm and ΔHm) and secondary and tertiary structures of the folded native proteins obtained from refolding of various denatured states (due to heat-, urea- and GdmCl-induced denaturation) of RNase-A in the presence of various osmolytes.

View Article: PubMed Central - PubMed

Affiliation: Dr. B. R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi, India.

ABSTRACT
Osmolytes are low molecular weight organic molecules accumulated by organisms to assist proper protein folding, and to provide protection to the structural integrity of proteins under denaturing stress conditions. It is known that osmolyte-induced protein folding is brought by unfavorable interaction of osmolytes with the denatured/unfolded states. The interaction of osmolyte with the native state does not significantly contribute to the osmolyte-induced protein folding. We have therefore investigated if different denatured states of a protein (generated by different denaturing agents) interact differently with the osmolytes to induce protein folding. We observed that osmolyte-assisted refolding of protein obtained from heat-induced denatured state produces native molecules with higher enzyme activity than those initiated from GdmCl- or urea-induced denatured state indicating that the structural property of the initial denatured state during refolding by osmolytes determines the catalytic efficiency of the folded protein molecule. These conclusions have been reached from the systematic measurements of enzymatic kinetic parameters (Km and kcat), thermodynamic stability (Tm and ΔHm) and secondary and tertiary structures of the folded native proteins obtained from refolding of various denatured states (due to heat-, urea- and GdmCl-induced denaturation) of RNase-A in the presence of various osmolytes.

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Secondary structural characteristic of the folded RNase-A at pH 7.0 and 25°C.Representative CD spectra of folded RNase-A (from heat-, GdmCl-, urea-induced denatured states) obtained from refolding in the presence of 1 M osmolytes. The CD spectra of the refolded RNase-A in the absence of osmolytes is identical with the native CD spectra and is omitted. Therefore, we have shown spectra only for the native (without refolding) control in this figure.
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pone-0109408-g004: Secondary structural characteristic of the folded RNase-A at pH 7.0 and 25°C.Representative CD spectra of folded RNase-A (from heat-, GdmCl-, urea-induced denatured states) obtained from refolding in the presence of 1 M osmolytes. The CD spectra of the refolded RNase-A in the absence of osmolytes is identical with the native CD spectra and is omitted. Therefore, we have shown spectra only for the native (without refolding) control in this figure.

Mentions: We have further investigated the native state structure of the folded proteins obtained from osmolyte-assisted refolding of different denatured states. Figure 4 and Figure 5 respectively show the effect of osmolytes on the secondary and tertiary structures of the refolded proteins. It is seen in these figures that both the secondary and tertiary structure of the osmolyte-induced folded native proteins obtained from the three different denatured states are identical. It is also seen in Figure S1 that there is no significant alteration in the environment of tyrosine of folded RNase-A obtained from the three different denatured states in presence of osmolytes. Figure 6 shows the heat-induced denaturation profiles of the folded proteins obtained from the osmolyte-assisted refolding of the different denatured states. The evaluated thermodynamic parameters (Tm and ΔHm) are presented in Table 3. It is seen in Figure 6 and Table 3 that at a given osmolyte concentration there is no significant difference in the thermodynamic stability (in terms of Tm and ΔHm) of the folded RNase-A obtained from osmolyte-assisted refolding of the three different denatured states. The results indicate that the observed increase in activity of the folded protein (in terms of decrease in Km and increase in kcat) obtained from the osmolyte-assisted refolding of the heat-induced denatured state relative to those obtained from refolding of the GdmCl- or urea-induced denatured state cannot be explained in terms of structure and stability of the characteristics of the folded protein.


Structural characteristic of the initial unfolded state on refolding determines catalytic efficiency of the folded protein in presence of osmolytes.

Warepam M, Sharma GS, Dar TA, Khan MK, Singh LR - PLoS ONE (2014)

Secondary structural characteristic of the folded RNase-A at pH 7.0 and 25°C.Representative CD spectra of folded RNase-A (from heat-, GdmCl-, urea-induced denatured states) obtained from refolding in the presence of 1 M osmolytes. The CD spectra of the refolded RNase-A in the absence of osmolytes is identical with the native CD spectra and is omitted. Therefore, we have shown spectra only for the native (without refolding) control in this figure.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4196897&req=5

pone-0109408-g004: Secondary structural characteristic of the folded RNase-A at pH 7.0 and 25°C.Representative CD spectra of folded RNase-A (from heat-, GdmCl-, urea-induced denatured states) obtained from refolding in the presence of 1 M osmolytes. The CD spectra of the refolded RNase-A in the absence of osmolytes is identical with the native CD spectra and is omitted. Therefore, we have shown spectra only for the native (without refolding) control in this figure.
Mentions: We have further investigated the native state structure of the folded proteins obtained from osmolyte-assisted refolding of different denatured states. Figure 4 and Figure 5 respectively show the effect of osmolytes on the secondary and tertiary structures of the refolded proteins. It is seen in these figures that both the secondary and tertiary structure of the osmolyte-induced folded native proteins obtained from the three different denatured states are identical. It is also seen in Figure S1 that there is no significant alteration in the environment of tyrosine of folded RNase-A obtained from the three different denatured states in presence of osmolytes. Figure 6 shows the heat-induced denaturation profiles of the folded proteins obtained from the osmolyte-assisted refolding of the different denatured states. The evaluated thermodynamic parameters (Tm and ΔHm) are presented in Table 3. It is seen in Figure 6 and Table 3 that at a given osmolyte concentration there is no significant difference in the thermodynamic stability (in terms of Tm and ΔHm) of the folded RNase-A obtained from osmolyte-assisted refolding of the three different denatured states. The results indicate that the observed increase in activity of the folded protein (in terms of decrease in Km and increase in kcat) obtained from the osmolyte-assisted refolding of the heat-induced denatured state relative to those obtained from refolding of the GdmCl- or urea-induced denatured state cannot be explained in terms of structure and stability of the characteristics of the folded protein.

Bottom Line: The interaction of osmolyte with the native state does not significantly contribute to the osmolyte-induced protein folding.We have therefore investigated if different denatured states of a protein (generated by different denaturing agents) interact differently with the osmolytes to induce protein folding.These conclusions have been reached from the systematic measurements of enzymatic kinetic parameters (Km and kcat), thermodynamic stability (Tm and ΔHm) and secondary and tertiary structures of the folded native proteins obtained from refolding of various denatured states (due to heat-, urea- and GdmCl-induced denaturation) of RNase-A in the presence of various osmolytes.

View Article: PubMed Central - PubMed

Affiliation: Dr. B. R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi, India.

ABSTRACT
Osmolytes are low molecular weight organic molecules accumulated by organisms to assist proper protein folding, and to provide protection to the structural integrity of proteins under denaturing stress conditions. It is known that osmolyte-induced protein folding is brought by unfavorable interaction of osmolytes with the denatured/unfolded states. The interaction of osmolyte with the native state does not significantly contribute to the osmolyte-induced protein folding. We have therefore investigated if different denatured states of a protein (generated by different denaturing agents) interact differently with the osmolytes to induce protein folding. We observed that osmolyte-assisted refolding of protein obtained from heat-induced denatured state produces native molecules with higher enzyme activity than those initiated from GdmCl- or urea-induced denatured state indicating that the structural property of the initial denatured state during refolding by osmolytes determines the catalytic efficiency of the folded protein molecule. These conclusions have been reached from the systematic measurements of enzymatic kinetic parameters (Km and kcat), thermodynamic stability (Tm and ΔHm) and secondary and tertiary structures of the folded native proteins obtained from refolding of various denatured states (due to heat-, urea- and GdmCl-induced denaturation) of RNase-A in the presence of various osmolytes.

Show MeSH
Related in: MedlinePlus