Unfolding Nature's Origami Acetonitrile's Role as a Protein Denaturant

Abstract

Protein function is determined not only by the structure of the folded, or native, conformation, but also by the stability and dynamics of the native state. To study folding, stability, and dynamics, denaturants such as Urea and GdmCl are used to perturb the native state, enabling detection of non-native conformations through spectroscopic methods. Linear extrapolation of multiple measurements as a function of denaturant concentration allows for extrapolation of ΔGNU to conditions in the absence of denaturant. The volatile solvent acetonitrile (AcN) is known to denature proteins but has not been systematically investigated. Here we validate the ability of acetonitrile to cooperatively denature two model proteins, myoglobin and trypsinogen, yielding ΔGNU values that closely match those obtained from traditional denaturants. In addition, we establish a method for accurately determining AcN concentration in denaturation samples, which is of critical importance given AcN's volatility. Together these results lay the foundation for use of AcN as a complementary denaturant that is compatible with hydrogen exchange mass spectrometry.