Spectroscopic evidence that osmolytes used in crystallization buffers inhibit a conformation change in a membrane protein.

BtuB is a bacterial outer-membrane protein that transports vitamin B(12). Spectroscopic studies using site-directed spin labeling (SDSL) indicate that the N-terminus of BtuB undergoes a dramatic structural change from a docked (folded) to an undocked (unfolded) configuration upon substrate binding. However, this dramatic conformational change is not observed in the crystal structures of BtuB. Here, we make an attempt to resolve this discrepancy and find that the effects of solutes can explain the discrepancy between the results obtained using these two methods. Specifically, if SDSL is performed with the buffers used for the crystallization of BtuB, the substrate-induced order-disorder transition of the N-terminal Ton box observed in intact membranes is blocked. Moreover, poly(ethylene glycol) 3350, which is a component of the crystallization and soaking buffers, is shown to inhibit this structural transition. It is likely that the crystal structure of BtuB in its holo form represents an osmotically trapped conformation. Conformational changes involving relatively modest energy differences and significant hydration changes may be sensitive to solutes used during crystallization, and this example demonstrates the value of combining multiple structural methods in the examination of protein structure and function.