Improvements to human health from the anticipated breakthroughs in proteomics will depend on a fuller understanding of unfolding/folding and dynamics of proteins and the effects of nonaqueous solvents on enzyme kinetics. Such understanding will be sought from studies of the model enzyme horseradish peroxidase (HRP). The temporal sequence of changes in secondary and tertiary structure during unfolding will be followed, changes in secondary and tertiary structure and enzyme activity induced by nonaqueous solvents will be correlated, and the dynamics for intraprotein diffusion of substrate analogues will be studied. Experimental methods will include fluorescence and phosphorescence, circular dichroism (CD), and UV/visible absorbance. Eventually, stopped-flow equipment will be sought to permit measurements of faster molecular events.
The specific aims are to test five hypotheses. 1) The temporal sequence of changes in secondary structure and tertiary structure during the unfolding of HRP can be determined by monitoring the relative time-course of the tryptophan fluorescence, near/UV CD, far/UV CD, and Soret CD absorbance. 2) Conformational changes in HRP in nonaqueous solutions can be measured and related to the significant reduction of enzyme activity. 3) Trifluoroethanol (TFE) alters the heme accessibility of HRP and alters its enzyme activity. 4) TFE alters the enzyme activity of HRP with other substrates. 5) The structure of substrates and substrate analogues for HRP can be correlated with the activation energy for their diffusion through the substrate channel of HRP.
| Schuh, Merlyn D; Baldwin, Melinda C (2006) Alpha-helix formation in melittin and beta-lactoglobulin A induced by fluorinated dialcohols. J Phys Chem B 110:10903-9 |
