Binding of bovine pancreatic trypsin inhibitor to trypsinogen: spectroscopic and volumetric studies.

Abstract

We have investigated the binding of bovine pancreatic trypsin inhibitor (BPTI) to bovine trypsinogen by combining ultrasonic velocimetry, high precision densimetry, and fluorescence spectroscopy. We report the changes in volume, adiabatic compressibility, van't Hoff enthalpy, entropy, and free energy that accompany the association of the two proteins at 25 degrees C and pH 8.0. We have used the measured changes in volume and compressibility in conjunction with available structural data to characterize the binding-induced changes in the hydration properties and intrinsic packing of the two proteins. Our estimate reveals that 110 +/- 40 water molecules become released to the bulk from the hydration shells of BPTI and trypsinogen. Furthermore, we find that the intrinsic coefficient of adiabatic compressibility of the two proteins decreases by 14 +/- 2%, which is suggestive of the binding-induced rigidification of the proteins' interior. BPTI-trypsinogen association is an entropy-driven event which proceeds with an unfavorable change in enthalpy. The favorable change in entropy results from partial compensation between two predominant terms. Namely, a large favorable change in hydrational entropy slightly prevails over a close in magnitude but opposite in sign change in configurational entropy. The reduction in configurational entropy and, consequently, protein dynamics is consistent with the observed decrease in intrinsic compressibility. In general, results of this work emphasize the vital role that water plays in modulating protein recognition events.