Conformational landscapes of rigid and flexible molecules explored with variable temperature ion mobility-mass spectrometry

Understanding the effect of temperature to the structural integrity and dynamics of proteins has relevance for many areas including biotechnology and the maintenance of a stable food supply for the climate emergency. The methods that can explore changes in structure as a function of sub-ambient temperature are scant, and yet many drugs are stored at such temperatures. Here we show how variable temperature ion mobility-mass spectrometry (VT-IM-MS) can provide the role of temperature on conformational landscapes in the form of collision cross sections at discrete temperatures. To delineate collision effects from structural change we report measurements made on four molecules that possess different degrees of rigidity namely: poly (L-lysine) (PLL) dendrimer, ubiquitin, β-casein and α-synuclein from 190-350K. We show that the PLL dendrimer varies with temperature consistent with collision theory, and conclude, as expected, its structure does not alter significantly over this range. By contrast, the structure of each protein is altered by the temperature of the drift gas, with notable unfolding to all charge states at 350 K and also at 250 K, following predicted in vitro stability curves, and with conformational variation that gives qualitative insights to the effect of temperature on the free energy landscape of these proteins. We also show that we can kinetically trap unfolding intermediates at drift temperatures of 210 K and 190 K on a millisecond experimental time scale. For alpha-synuclein, the 13+ ions present two distinct conformers and VT-IM-MS measurements allow us to calculate the transition rate and activation energies for conversion between these. These data exemplify the capability of VT-IM-MS to provide insights to thermodynamics involved in conformational restructuring.