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Comparison of physico-chemical aspects between E. coli and human dihydrofolate reductase: an equilibrium unfolding study

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Abstract

A protein, differing in origin, may exhibits variable physico-chemical behaviour, difference in sequence homology, fold and function. Thus studying structure-function correlationship of proteins from altered sources is meaningful in the sense that it may give rise to comparative aspects of their sequence−structure−function correlationship. Dihydrofolate reductase is an enzyme involved in cell cycle regulation. It is a significant enzyme targeted to develop anticancer drugs. Hence, detailed understanding of structurefunction relationships of wide variants of DHFR enzyme would be important for developing inhibitor or an antagonist against the enzyme involved in the cellular developmental processes. In this communication, we have reported the comparative structure-function relationship between E. coli and Human DHFR. The differences in the unfolding behaviour of these two proteins have been investigated to understand various properties like differences in relative stability and variation in conformational changes between them under identical denaturation conditions. The equilibrium unfolding mechanism of DHFR proteins using GdnHCl as denaturant in the presence of various types of osmolytes has been monitored using loss in enzymatic activity, changes in intrinsic tryptophan fluorescence and extrinsic ANS bound fluorescence. It has been observed that osmolytes, such as 1 M sucrose, and 30% glycerol, provided enhanced stability to both the variants of DHFR. Their level of stabilisation has been observed to be dependent on intrinsic protein stability. It was observed that 100 mM proline does not show any significant stabilization to either DHFRs. In the present study, it has been observed that the human protein is relatively less stable than the E. coli counterpart.

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Correspondence to Pratima Chaudhuri (Chattopadhyay).

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Published in Russian in Biofizika, 2015, Vol. 60, No. 3, pp. 471–480.

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Thapliyal, C., Jain, N. & Chaudhuri (Chattopadhyay), P. Comparison of physico-chemical aspects between E. coli and human dihydrofolate reductase: an equilibrium unfolding study. BIOPHYSICS 60, 378–386 (2015). https://doi.org/10.1134/S0006350915030197

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  • DOI: https://doi.org/10.1134/S0006350915030197

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