Abstract

The reversible thermal unfolding of wild type and mutant dihydrofolate reductase (DHFR) from E. coli has been previously studied by optical spectroscopy. This information indicated the presence of molten globule type intermediates as well as irreversible type melting. In order to further explore these results, a series of DSC experiments at a number of different pHs were conducted using the C85S/C152E DHFR mutant. The lack of the disulfide bonds, that are present in the wild-type, greatly increased the reversibility of the thermal denaturation. It was also found that the protein achieves maximum stability at pH 7.0 with destabilization occurring above and below this value. Presently, experiments are being conducted at high salt concentrations in an attempt to further stabilize possible intermediates. Circular dichroism (CD) at reduced concentrations will also be performed to investigate the concentration dependent behavior found in the DSC results.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR004328-09
Application #
5224886
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
9
Fiscal Year
1996
Total Cost
Indirect Cost

  Publications

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Zakharov, S D; Lindeberg, M; Griko, Y et al. (1998) Membrane-bound state of the colicin E1 channel domain as an extended two-dimensional helical array. Proc Natl Acad Sci U S A 95:4282-7
Luque, I; Todd, M J; Gomez, J et al. (1998) Molecular basis of resistance to HIV-1 protease inhibition: a plausible hypothesis. Biochemistry 37:5791-7
Chu, V; Freitag, S; Le Trong, I et al. (1998) Thermodynamic and structural consequences of flexible loop deletion by circular permutation in the streptavidin-biotin system. Protein Sci 7:848-59

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