We used time-dependent fluorescence energy transfer to determine the distribution of donor-to-acceptor distances in native and denatured troponin I(TnI). The single tryptophan residue (trp 158) of TnI served as the donor (D), and the acceptor (A) was a labeled cysteine residue (cys 133). The time-dependent intensity decays of the donor were measured by the frequency-domain method. The frequency-response of the donor emission, in the absence and presence of acceptor, was used to recover the distribution of D to Å distances, using an algorithm which accounts for the intrinsic multi-exponential decay of the donor. In the native state the D-A distribution is characterized by an average distance of 23 A and a half-width of 12 Å. Denaturation results in a modest increase in the average distance to 27 A, and a dramatic increase in half-width to 47 Å. We believe the ability to recover distance distributions will have numerous applications in the characterization of biological macromolecules.
|Number of pages||7|
|Journal||Proceedings of SPIE - The International Society for Optical Engineering|
|State||Published - 24 Jun 1988|