Resolution of end-to-end diffusion coefficients and distance distributions of flexible molecules using fluorescent donor-acceptor and donor-quencher pairs.
We used time-dependent fluorescence energy transfer, time-dependent collisional quenching, and global analysis of the data resulting from these through-space and contact interactions to recover the end-to-end distance distributions and diffusion coefficients of flexible fluorescent molecules. The fluorescence decays of covalently linked tryptamine-acceptor and tryptamine-quencher pairs were measured by the frequency-domain method. These data were fit using numerical solutions of the differential equation, which predicts the time- and distance-dependent population of the excited state donors in the presence of energy transfer or collisional quenching, followed by transformation to the frequency domain for nonlinear least-squares comparison with the experimental data. We found that the energy transfer data for the donor-acceptor pair alone were adequate to recover the starting distribution and the end-to-end diffusion coefficient; however, the resolution is dramatically improved by the use of both the through-space and contact interactions.
Lakowicz
J R
JR
Department of Biological Chemistry, School of Medicine, University of Maryland, Baltimore 21201.
Kuśba
J
J
Szmacinski
H
H
Gryczynski
I
I
Eis
P S
PS
Wiczk
W
W
Johnson
M L
ML
D008433:Mathematics; D008962:Models, Theoretical; D008968:Molecular Conformation; D013050:Spectrometry, Fluorescence / Q000379:methods