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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 various denatured states of RNase-A at pH 7.0.The GdmCl- and urea- induced denatured states were generated using 6.5 M GdmCl and 8.5 M urea respectively. The heat induced-denatured state was generated by incubating the protein at 85°C for 15 minutes.
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pone-0109408-g001: Secondary structural characteristic of various denatured states of RNase-A at pH 7.0.The GdmCl- and urea- induced denatured states were generated using 6.5 M GdmCl and 8.5 M urea respectively. The heat induced-denatured state was generated by incubating the protein at 85°C for 15 minutes.

Mentions: Heat, GdmCl or urea has earlier been known to induce different denatured states having different structural properties. Heat-induced denatured state retains a large amount of residual secondary structures while GdmCl or urea induces a random coil denatured conformation [25], [26], [27]. To investigate the effect of different osmolytes (proline, glycine, β-alanine, TMAO, sarcosine, betaine) on protein folding, we have chosen RNase-A and generated its different denatured states using different denaturing agents (heat, GdmCl, urea) (see Figure 1). It is clearly seen in this figure that heat-induced denatured state is different from that of either GdmCl- or urea-induced denatured state. Table 1 shows the measured enzymatic kinetic parameters (Km and kcat) of the folded proteins obtained from refolding of the respective heat-, GdmCl- or urea-induced denatured state. We have compared the change in Km and kcat of refolded proteins obtained from the three different denatured states in the presence of osmolytes by plotting ΔKm versus osmolyte concentration (see Figure 2)and Δkcat versus osmolyte concentration (see Figure 3). It is seen in these figures that the activity of the folded protein obtained from osmolyte-assisted refolding of heat-induced denatured state has a decreased Km and an increased kcat of the RNase-A-mediated hydrolysis of cytidine 2′–3′ cyclic mono phosphate (c>p). On the other hand, Km and kcat values of the folded proteins obtained from the osmolyte-assisted refolding of GdmCl- or urea-induced denatured state remain unchanged (except in case of TMAO). Furthermore, Table 2 shows the measured Km and kcat values of the native RNase-A in presence of 1 M osmolytes.


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 various denatured states of RNase-A at pH 7.0.The GdmCl- and urea- induced denatured states were generated using 6.5 M GdmCl and 8.5 M urea respectively. The heat induced-denatured state was generated by incubating the protein at 85°C for 15 minutes.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0109408-g001: Secondary structural characteristic of various denatured states of RNase-A at pH 7.0.The GdmCl- and urea- induced denatured states were generated using 6.5 M GdmCl and 8.5 M urea respectively. The heat induced-denatured state was generated by incubating the protein at 85°C for 15 minutes.
Mentions: Heat, GdmCl or urea has earlier been known to induce different denatured states having different structural properties. Heat-induced denatured state retains a large amount of residual secondary structures while GdmCl or urea induces a random coil denatured conformation [25], [26], [27]. To investigate the effect of different osmolytes (proline, glycine, β-alanine, TMAO, sarcosine, betaine) on protein folding, we have chosen RNase-A and generated its different denatured states using different denaturing agents (heat, GdmCl, urea) (see Figure 1). It is clearly seen in this figure that heat-induced denatured state is different from that of either GdmCl- or urea-induced denatured state. Table 1 shows the measured enzymatic kinetic parameters (Km and kcat) of the folded proteins obtained from refolding of the respective heat-, GdmCl- or urea-induced denatured state. We have compared the change in Km and kcat of refolded proteins obtained from the three different denatured states in the presence of osmolytes by plotting ΔKm versus osmolyte concentration (see Figure 2)and Δkcat versus osmolyte concentration (see Figure 3). It is seen in these figures that the activity of the folded protein obtained from osmolyte-assisted refolding of heat-induced denatured state has a decreased Km and an increased kcat of the RNase-A-mediated hydrolysis of cytidine 2′–3′ cyclic mono phosphate (c>p). On the other hand, Km and kcat values of the folded proteins obtained from the osmolyte-assisted refolding of GdmCl- or urea-induced denatured state remain unchanged (except in case of TMAO). Furthermore, Table 2 shows the measured Km and kcat values of the native RNase-A in presence of 1 M osmolytes.

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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Related in: MedlinePlus