Biophysical characterization of α-amylase inhibitor Parvulustat (Z-2685) and comparison with Tendamistat (HOE-467)

Parvulustat is a small, highly active proteinaceous α-amylase inhibitor whose high-resolution NMR structure was recently solved in Frankfurt. Here, we present its biochemical and biophysical characterization. Several spectroscopic methods such as UV, fluorescence and CD were utilized to extract conformational changes upon modification of pH, temperature and chemical denaturant. Parvulustat revealed native like behavior over a wide range of denaturizing agents as reflected in terms of activity and thermodynamic data. In addition, spectroscopic and thermodynamic properties of Parvulustat were compared to the well-characterized Tendamistat. Despite the overall structural similarity, the thermodynamic stability of the two proteins is different. Our analysis led to the conclusion that Parvulustat is even more stable than Tendamistat. Furthermore, investigations on three C-terminally truncated Parvulustat derivatives indicate that the higher stability is caused by the long flexible C-terminus.

► Parvulustat is a very potent-amylase inhibitor (Ki 2.8x10-11 M) over a wide range of pH.
► Native like structure is found for denaturizing agents aslow pH and high temperature.
► Its very high stability is mostly caused by the c-terminus and hydrophobic core.
► Parvulustat is an excellent model protein for studies on oxidative folding of-sheet proteins.