Structure-activity correlationship and folding of recombinant Escherichia coli dihydro folate reductase (DHFR) enzyme through biochemical and biophysical approaches
Jai Mittal, Gayathri Ravitchandirane, Tapan K. Chaudhuri, Pratima Chaudhuri
DOI: 10.4236/jbpc.2010.12013   PDF    HTML     6,066 Downloads   10,505 Views   Citations


The design of any antagonist or inhibitor for any enzyme requires the knowledge of structure- function relationship of the protein and the optimum conformational states for maximum and minimum activities. Furthermore, designing of the inhibitors or drugs against an enzyme becomes easier if there is information available about various well characterized intermediate conformation of the molecule. In vivo folding pathway of any recombinant protein is an important parameter for understanding its ability to fold by itself inside the cell, which always dictates the downstream processing for the purification. In the present manuscript we have discussed about the in vivo and in vitro folding, and structure-function relationship of Dihydrofolate reductase enzyme. This is an important enzyme involved in the cell growth and hence inhibition or inactivation of the enzyme may reduce the cell growth. It was observed that the equilibrium unfolding transition of DHFR proceeds through the formation of intermediates having higher exposed surface hydrophobicity, unchanged enzymatic activity and minimum changes in the secondary structural elements. Because of enhanced surface hydrophobicity, and unchanged enzymatic activity, these intermediates could be a nice target for designing drugs against DHFR.

Share and Cite:

Mittal, J. , Ravitchandirane, G. , Chaudhuri, T. and Chaudhuri, P. (2010) Structure-activity correlationship and folding of recombinant Escherichia coli dihydro folate reductase (DHFR) enzyme through biochemical and biophysical approaches. Journal of Biophysical Chemistry, 1, 105-112. doi: 10.4236/jbpc.2010.12013.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Dobson, C.M. (2001) The structural basis of protein folding and its links with human disease. Philosophical Transactions of the Royal Society of London, Series B, Biological Sciences, 356(1406), 133-145.
[2] Aiso, K., Nozaki, T., Shimoda, M. and Kokue, E. (1999) Assay of dihydrofolate reductase activity by monitoring tetrahydrofolate using high-performance liquid chromatography with electrochemical detection. Analytical Biochemistry, 272(2), 143-148.
[3] Sambrook, J. and Russell, D. (2001) Molecular cloning: A laboratory manual. 3rd Edition, Cold Spring Harbor Laboratory Press, New York, A8, 1-55.
[4] Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(5259), 680-685.
[5] Chaudhuri, T.K., Farr, G.W., Fenton, W.A., Rospert, S. and Horwich, A.L. (2001) GroEL/GroES-mediated folding of a protein too large to be encapsulated. Cell, 107(2), 235-246.
[6] Rao, K.N. and Venkatachalam, S.R. (2000) Inhibition of dihydrofolate reductase and cell growth activity by the phenanthroindolizidine alkaloids pergularinine and tylophorinidine: The in vitro cytotoxicity of these plant alkaloids and their potential as antimicrobial and anticancer agents. Toxicology in Vitro, 14(1), 53-59.
[7] Hillcoat, B.L., Nixon, P.F. and Blakley, R.L. (1967) Effect of substrate decomposition on the spectrophotometric assay of dihydrofolate reductase. Analytical Biochemistry, 21(2), 178-189.
[8] Yang, J.T., Wu, C.-S.C. and Martinez, H.M. (1986) Calculation of protein conformation from circular dichroism. Methods in Enzymology, 130, 208-269.

Copyright © 2024 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.