Effect of proline to alanine mutation on the thermal stability of the all-β-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 Tm was found as 82.3±0.5 °C in 2H2O. The thermal stability of the proline-free mutant is reduced by 15 °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. [Copyright &y& Elsevier]