Protein Unfolding in Freeze Frames: Intermediates of Ubiquitin and Lysozyme Revealed by Variable Temperature Ion Mobility-Mass Spectrometry

As experimentalists, we normally rely on assessing observables. However sometimes, the most fascinating phenomena are not noticeable directly. An example of such is our data and the corresponding interpretation presented in this manuscript. We have designed and constructed a ion mobility mass spectrometer (acs.analchem.6b01812) capable of taking ion mobility measurements over a temperature range from 150-500K. We chose to benchmark this new instrument, using the small proteins Ubiquitin and Lysozyme extensively studied as a “model proteins” in many in-silico, -solution and -vacuo studies focusing on conformational dynamics.In this work, we activate and subsequently thermally equilibrate the protein ions at several temperatures prior to collision cross section measurement. For Ubiquitin at 300K and above, the protein unfolds in a “step-wise” fashion as previously reported (by David Clemmer) and for other proteins including lysozyme, and cytochrome c by us and also by Martin Jarrold. However, to our surprise, activation and equilibration of ubiquitin at 150K yields a plethora of highly extended forms of the protein. We attribute these as kinetically trapped unfolding intermediates. Since the activation process is the same at both temperatures we infer that the unfolding must always proceed via these extended intermediate forms, which then converge to commonly reported conformations. Intriguingly, this “convergence” appears to occur mostly below the temperature of irreversible conformational thermal transition of Ubiquitin reported in many solution phase studies. For Lysozyme the same experiment is performed and similar results are obtained although we cannot activate the gaseous ensemble too far from the native fold and the activation barrier to refolding is low enough to allow it to be re-accessed on the experimental timescale we use.