Physics Colloquium
Friday, January 22nd, 2004, 4:00 P.M.

Gail E. Fanucci

Dept. of Chemistry
University of Virginia

Site-Directed Spin Labeling and
EPR spectroscopy of the outer membrane
Vitamin B12 Transporter BtuB

BtuB is a 66kD bacterial outer-membrane protein that sequesters vitamin B12 from the surroundings and transports it into the periplasm. Site-directed spin labeling (SDSL) and EPR spectroscopy are used to investigate the effects of substrate binding and sample preparation conditions on the conformation of the N-terminal energy coupling Ton box motif. Examples of substrates include Ca2+, Ca2+/B12 and colicin E3 binding domains. The effects of sample conditions such as different membrane mimetic environments and osmolytes are discussed. Recently, crystal structures of BtuB with various substrates have been reported. Here we compare our spectroscopic EPR results of BtuB to the reported crystal structures. In particular, we contrast the different models for substrate-induced conformational changes in the Ton box region of BtuB. EPR investigations indicate that a substrate-induced order-disorder transition occurs in the N-terminal region of BtuB. On the other hand, the X-ray model shows a much subtler conformational change upon substrate binding. However, when EPR is performed on spin-labeled BtuB samples in the buffers used during crystalization, the order-disorder transition is blocked. The crystallization buffers typically include salts and polyethylene glycols as precipitating agents, and here we demonstrate that PEG 3350 and LiCl2 can inhibit the conformational change that is observed in earlier EPR investigations. Hence, it is likely that the crystal structure of the Ca2+/B12 bound state of BtuB represents an "osmotically trapped" conformation.