Identification of the Escherichia Coli SecA Solution State Dimer Orientation using Forster Resonance Energy Transfer

Abstract

Over the past 8 years four different SecA dimer crystal structures have been solved. While the subunits of the dimer remain essentially the same, the orientation of the subunits is drastically different for each of the crystal structures. Some data has been published (Ding, H., Hunt, J.F., Mukerji, I., Oliver, D.B. 2003 Biochemistry 42, 8729-38) that suggest one of the anti-parallel dimers (Hunt, J.F., Weinkauf, S., Henry, L., Fak, J.J., McNicholas, P., Oliver, D.B., Deisenhofer, J. 2002 Science 297, 2018-26) is the solution state orientation, while other data suggests that it is the parallel dimer (Vassylyev, D.G., Mori, H., Vassylyeva, M.N., Tsukazaki, T., Kimura, Y., Tahirov, T.H., Ito, K. 2006 J Mol Biol 364, 248-58). Thus, the solution state orientation of the SecA dimer is currently unknown. The aim of the present study is to identify the dominant dimer orientation of E. coli SecA in solution. Forster Resonance Energy Transfer (FRET) was used to measure distances between dye-labeled monocysteine residues on SecA dimer subunits. Three fluorescent dye pairs with R0 values of 34 A, 62 A, and 82 A were used to measure distances from ∼11 A to 140 A, which corresponds to the possible distances expected within any of the 4 proposed dimer orientations. The FRET measured distances were then compared to those measured in the SecA dimer crystal structures to determine if one of the structures represents the dominant solution state dimer orientation or if a mixed population of dimer states appears likely.Funding provided by National Institute of Health