Effect of proline to alanine mutation on the thermal stability of the all-beta-sheet protein tendamistat.

The temperature dependent denaturation of wild-type tendamistat and of the proline-free triple mutant P7A/P9A/P50A was investigated using Fourier-transform infrared (FTIR) spectroscopy. Whereas the temperature-induced unfolding is reversible in the wild type, aggregation was observed for the proline-free tendamistat when studied under the same conditions. The midpoint unfolding temperature T(m) was found as 82.3+/-0.5 degrees C in (2)H2O. The thermal stability of the proline-free mutant is reduced by 15 degrees C as compared to the wild type. Changes in the strength of hydrogen bonding of tyrosine O-H groups upon unfolding and aggregation are reflected in small shifts of the C-C stretching mode of the aromatic ring near 1515 cm(-1). Evaluation of data from different infrared (IR) bands sensitive to changes in secondary structure as well as to changes in tertiary structure strongly supports a two-state model for the unfolding process of wild-type tendamistat.