Proteins that fold with assistance populate a “folding landscape” that is shaped by the chaperones or catalysts that facilitate their folding. Those proteins that function independently after folding with assistance experience a different “native landscape”, determined only by their sequence. The absence of the pressure to fold may have allowed these proteins to evolve their native landscape for function more optimally than those that spontaneously fold. Indeed, our previous work on alpha-lytic protease (αLP), which folds with the assistance of an intramolecular folding catalyst, provided the first demonstration that the native, folded state of a protein can be less thermodynamically stable than unfolded states (Sohl, Jaswal, et. al. Nature 395, 817-819, 1998). Furthermore, αLP’s unique mechanism of stabilizing its native state through a large, highly cooperative barrier to unfolding (kinetic stability) leads to highly optimized functional longevity: αLP survives proteolytic attack one hundred-fold longer than its spontaneously folding homologues (Jaswal, et. al., Nature 415, 343-436, 2002). Therefore the native landscapes of other proteins that fold with assistance may also have very different features and reveal novel strategies for stability and function.
We are using our HXMS approach in combination with proteolytic sensitivity, and traditional spectroscopic measurements to identify and explore diverse mechanisms of stability. We are investigating the determinants of kinetic stability by mapping the landscapes of a spectrum of aLP homologues and mutants varying in function, assessed by enzymatic activity and longevity. To test the hypothesis that the evolution of functionally important unique native landscape characteristics may be a general feature of many “folding-challenged” proteins, we are mapping the landscapes of other proteins that fold with catalysts, amyloid precursor proteins, and chaperone substrates. Finally, we are exploiting our HXMS approach to rapidly map the landscapes of small spontaneously folding proteins as a function of temperature, in order to provide a more complete basis set for understanding the difference in the physical chemical origins of stability through kinetics, as in αLP, or other non-traditional mechanisms that come to light as we explore more landscapes of “folding-challenged” proteins.