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# Thermodynamic Effects of Proline Introduction on Protein Stability

Prajapati, Ravindra Singh and Das, Mili and Sreeramulu, Sridhar and Sirajuddin, Minhajuddin and Srinivasan, Sankaranarayanan and Krishnamurthy, Vaishnavi and Ranjani, Ranganathan and Ramakrishnan, C and Varadarajan, Raghavan (2007) Thermodynamic Effects of Proline Introduction on Protein Stability. In: Proteins: Structure, Function, and Bioinformatics, 66 (2). pp. 480-491.

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## Abstract

The amino acid Pro is more rigid than other naturally occurring amino acids and, in proteins, lacks an amide hydrogen. To understand the structural and thermodynamic effects of Pro substitutions, it was introduced at 13 different positions in four different proteins, leucine–isoleucine–valine binding protein, maltose binding protein, ribose binding protein, and thioredoxin. Three of the maltose binding protein mutants were characterized by X-ray crystallography to confirm that no structural changes had occurred upon mutation. In the remaining cases, fluorescence and CD spectroscopy were used to show the absence of structural change. Stabilities of wild type and mutant proteins were characterized by chemical denaturation at neutral pH and by differential scanning calorimetry as a function of pH. The mutants did not show enhanced stability with respect to chemical denaturation at room temperature. However, 6 of the 13 single mutants showed a small but significant increase in the free energy of thermal unfolding in the range of 0.3–2.4 kcal/mol, 2 mutants showed no change, and 5 were destabilized. In five of the six cases, the stabilization was because of reduced entropy of unfolding. However, the magnitude of the reduction in entropy of unfolding was typically several fold larger than the theoretical estimate of $-4\hspace{2mm}cal\hspace{2mm} K^{-1} mol^{-1}$ derived from the relative areas in the Ramachandran map accessible to Pro and Ala residues, respectively. Two double mutants were constructed. In both cases, the effects of the single mutations on the free energy of thermal unfolding were nonadditive.

Item Type: Journal Article Copyright of this article belongs to Wiley InterScience. Thermostability; Protein engineering; Entropy; Unfolded state; conformational flexibility Division of Biological Sciences > Molecular Biophysics Unit 12 Mar 2007 19 Sep 2010 04:34 http://eprints.iisc.ernet.in/id/eprint/9693

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