Mapping the Conformational Landscape of a Dynamic Enzyme

33 Determining the interconverting conformations of dynamic proteins in atomic detail is a major challenge for 34 structural biology. Conformational heterogeneity in the active site of the dynamic enzyme cyclophilin A (CypA) 35 has been previously linked to its catalytic function, but the extent to which the different conformations of these 36 residues are correlated is unclear. Here we compare the conformational ensembles of CypA by multitemperature 37 synchrotron crystallography and fixed-target X-ray free electron laser (XFEL) crystallography. The “diffraction38 before-destruction” nature of XFEL experiments provides a radiation-damage-free view of the functionally 39 important alternative conformations of CypA, confirming earlier synchrotron-based results. We monitored the 40 temperature dependences of these alternative conformations with eight synchrotron datasets spanning 100-310 K. 41 Multiconformer models show that many alternative conformations in CypA are populated only at 240 K and 42 above, yet others remain populated or become populated at 180 K and below. These results point to a complex 43 evolution of conformational heterogeneity between 180-240 K that involves both thermal deactivation and 44 solvent-driven arrest of protein motions in the crystal. The lack of a single shared conformational response to 45 temperature within the dynamic active-site network provides evidence for a conformation shuffling model, in 46 which exchange between rotamer states for a large aromatic ring in the middle of the network shifts the 47 conformational ensemble for the other residues in the network. Together, our multitemperature analyses and 48 XFEL data motivate a new generation of temperatureand time-resolved experiments to structurally characterize 49 the dynamic underpinnings of protein function. 50 . CC-BY 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/016733 doi: bioRxiv preprint first posted online Mar. 19, 2015;