Monitoring structural stability of trypsin inhibitor at the submolecular level by amide-proton exchange using Fourier transform infrared spectroscopy: a test case for more general application.

Combining the information on the secondary structure content as present in the shape of a protein amide I infrared band with the approach of monitoring amide-proton exchange using infrared spectroscopy, we have been able to investigate the structural stability of different components present in a protein, which are shown to be correlated to the different classes of secondary structures. For this purpose, the changes in intensity in different regions of the amide I have been detected upon exposure of the protein to a 2H2O environment, revealing four separate classes of exchanging components. As a test case for the approach described in this work, the amide-proton exchange of hydrated protein films of bovine pancreatic trypsin inhibitor has been studied using infrared spectroscopy, and is compared to literature data obtained by other techniques. A slow amide-proton exchange is observed for a class correlated to the beta-strands present in the protein, with protection of amide-protons for more than 19 h. Another class, which has been assigned to mainly helical residues, shows much less protection from exchange. The distribution function of the exchange rates of a class linked to the beta-turns displays five times faster exchange rates compared to those found for the majority of the helical residues, but they are still ten times slower compared to a class which we defined to represent the nonstructured parts of the protein.