Project Summary: The goal of this project is to understand how enzymes utilize conformational fluctuations in particular, local unfolding, to facilitate catalysis. Over the past several decades it has become increasingly clear that rather existing as static structures, proteins are actually ensembles of sometimes very different conformational states, and the fluctuations are critical to function. It is of great import to know how this is done. Are there unifying principles that connect proteins with different functions? Here we take advantage of several key discoveries discoveries by our group during the previous funding cycles, which demonstrates that the enzyme adenylate kinase (AK) from E. coli, uses local unfolding to modulate its enzymatic activity – in effect, the energy landscape has unfolding within its functionally important repertoire. We found unfolding to occur in both the LID and the AMPbd domains and that unfolding in the different regions selectively modulated different key enzymatic parameters, with changes in one lid modulating Km , and changes in the other modulating kcat. Unexpectedly we found that local unfolding actually controlled cold adaptation in the enzyme, thus directly demonstrating the functional importance. Our discovery stands in stark contrast to the current accepted model (which posits a rigid-body opening and closing reaction facilitated by a hinge that is believed to facilitate catalytic turnover). The fact that AK is representative of more than 3,000 high-resolution structures in the Protein Data Bank )PDB) that have been hypothesized (but never actually demonstrated) to utilize the rigid-body open/closing motions to facilitate catalysis, suggests that order/disorder fluctuations may be more prevalent than previously believed. How general is unfolding and how does its presence impact the more than 40 years of structural biology-based functional studies? Our approach is two-fold. First, as our results directly undermine the existing models of AK (and thus require a new model) we will determine how the disordered states discovered by us are responsible for the function of the enzyme. Second, we must interrogate the database of enzymes to determine how general local unfolding and disorder are. Do all enzymes utilize unfolding? Can we develop a quantitative, experimentally-derived model of AK and other enzymes? We will perform binding and stability measurements using isothermal titration calorimetry (ITC), circular dichroism (CD) monitored thermal unfolding and hydrogen exchange (HX), and we will monitor the kinetics of the conformational and enzymatic processes using NMR CEST (conformational exchange saturation transfer) and steady state enzymatic analysis.