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An improved experimental system for determining small folding entropy changes resulting from proline to alanine
Timothy O Street1, Christina Marchetti Bradley, Doug Barrick
1T.C. Jenkins Department of Biophysics, Johns Hopkins University, 204 Jenkins Hall, 3400 N. Charles Street, Baltimore, MD 21218, USA.
Protein Science : a Publication of the Protein Society
|September 1, 2005
Summary
Proline to alanine substitutions increase protein unfolding entropy. This study quantifies this entropic destabilization in a repeat protein, confirming predictions and providing experimental values for protein stability.
Area of Science:
- Protein biochemistry
- Biophysical chemistry
- Molecular biology
Background:
- Protein stability is influenced by the conformational entropy of the unfolded state.
- Proline to alanine substitutions were hypothesized to increase unfolding entropy without affecting folded state entropy.
- Quantifying these entropic changes experimentally has been challenging.
Purpose of the Study:
- To experimentally quantify the unfolding entropy change associated with proline to alanine substitutions.
- To validate the hypothesis that proline to alanine substitutions lead to entropic destabilization.
- To investigate the effect of multiple proline to alanine substitutions on unfolding entropy.
Main Methods:
- Construction of protein variants with multiple proline to alanine substitutions in the Drosophila Notch ankyrin domain.
- Determination of unfolding entropy values using chemical denaturation and free energy measurements over a temperature range.
- Analysis of structural data to compare alanine and proline residue conformations.
Main Results:
- Increasing proline residues in similar environments correlates with increased unfolding entropy.
- The average measured increase in unfolding entropy for proline to alanine substitutions is 7.7 +/- 4.2 cal mol(-1) K(-1).
- Experimental results are consistent with the initial estimations by Matthews et al. (1987).
Conclusions:
- Proline to alanine substitutions effectively increase protein unfolding entropy, leading to entropic destabilization.
- The experimental quantification supports the theoretical prediction of entropic contributions to protein stability changes.
- This work provides a method for accurately measuring entropic changes due to specific amino acid substitutions.