| Abstract
| - BtuB is a bacterial outer-membrane protein that transports vitamin B12. Spectroscopic studiesusing site-directed spin labeling (SDSL) indicate that the N-terminus of BtuB undergoes a dramatic structuralchange 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 anattempt to resolve this discrepancy and find that the effects of solutes can explain the discrepancy betweenthe results obtained using these two methods. Specifically, if SDSL is performed with the buffers usedfor the crystallization of BtuB, the substrate-induced order−disorder transition of the N-terminal Ton boxobserved in intact membranes is blocked. Moreover, poly(ethylene glycol) 3350, which is a componentof the crystallization and soaking buffers, is shown to inhibit this structural transition. It is likely that thecrystal structure of BtuB in its holo form represents an osmotically trapped conformation. Conformationalchanges involving relatively modest energy differences and significant hydration changes may be sensitiveto solutes used during crystallization, and this example demonstrates the value of combining multiplestructural methods in the examination of protein structure and function.
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