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. 1988 Oct;54(4):577–586. doi: 10.1016/S0006-3495(88)82992-8

Resolution of end-to-end distance distributions of flexible molecules using quenching-induced variations of the Forster distance for fluorescence energy transfer.

I Gryczynski 1, W Wiczk 1, M L Johnson 1, H C Cheung 1, C K Wang 1, J R Lakowicz 1
PMCID: PMC1330361  PMID: 3224143

Abstract

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.

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Selected References

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