Resolution of end-to-end distance distributions of flexible molecules using quenching-induced variations of the Forster distance for fluorescence energy transfer.
We describe a new method to recover the distribution of donor-to-acceptor (D-A) distances in flexible molecules using steady-state measurements of the efficiency of fluorescence energy transfer. The method depends upon changes in the Forster distance (Ro) induced by collisional quenching of the donor emission. The Ro-dependent transfer efficiencies are analyzed using nonlinear least squares to recover the mean D-A distance and the width of the distribution. The method was developed and tested using three synthetic D-A pairs, in which the chromophores were separated by alkyl chains of varying lengths. As an example application we also recovered the distribution of distances from the single tryptophan residue in troponin I (trp 158) to acceptor-labeled cysteine 133. The half-width of the distribution increases from 12 A in the native state to 53 A when unfolded by guanidine hydrochloride. For both TnI and the three model compounds the distance distributions recovered from the steady-state transfer efficiencies were in excellent agreement with the distributions recovered using the more sophisticated frequency-domain method (Lakowicz, J.R., M.L. Johnson, W. Wiczk, A. Bhat, and R.F. Steiner. 1987. Chem. Phys. Lett. 138:587-593). The method was found to be reliable and should be generally useful for studies of conformational distributions of macromolecules.
Gryczynski
I
I
University of Maryland, School of Medicine, Department of Biological Chemistry, Baltimore 21201.
Wiczk
W
W
Johnson
M L
ML
Cheung
H C
HC
Wang
C K
CK
Lakowicz
J R
JR
D004735:Energy Transfer; D005230:Fatty Acids, Nonesterified; D008962:Models, Theoretical; D008968:Molecular Conformation; D015394:Molecular Structure; D011789:Quantum Theory; D013050:Spectrometry, Fluorescence / Q000379:methods; D014363:Tryptamines
D005230:Fatty Acids, Nonesterified; D014363:Tryptamines; C030820:tryptamine