End-to-end diffusion on the microsecond timescale measured with resonance energy transfer from a long-lifetime rhenium metal-ligand complex

Abstract

We measured the end-to-end diffusion coefficient of an alkyl chain-linked donor-acceptor pair using the time-resolved frequency-domain decay of the donor. The donor was a rhenium metal-ligand complex with a mean decay time ranging from 2.1 to 7.9 microseconds in the absence of the Texas red acceptor. The decay time was used to measure the donor-to-acceptor distance distribution and the mutual diffusion coefficient. Using this long lifetime donor, it was easily possible to determine a diffusion coefficient near 2 x 10(-8) cm2/s and diffusion coefficients as low as 1.3 x 10(-9) cm2/s were measurable. Such long lifetime donors should be valuable for measuring the flexing of peptides on the microsecond timescale, domain motions of proteins and lateral diffusion in membranes. The availability of microsecond decay time luminophores now allows luminescence spectroscopy to be useful generally for studies of microsecond dynamics of biological macromolecules.

Figure 1.

Spectral overlap of the Re-MLC donor emission (— — —) with the Tr acceptor absorption (—). The lower panel shows the emission spectra of the Re-MLC donor (— — —), the Tr acceptor (—•• —•• —) and of the covalently linked D–A pair (——). In this D–A pair the donor emission at 530 nm is quenched and there is a small enhancement of the acceptor emission. All spectra are in propylene glycol at 20°C.

Figure 2.

Frequency-domain intensity decays of the Re-MLc donor(····) and of the D–A pair (larger dots) in propylene glycol at −20 (top) and 20°C (bottom). The data points for the Re-MLC donor alone decay are not shown but the multiexponential analyses are shown in Table 1. The solid lines shows the best fit using the distance distribution model with diffusion (Table 1). The dashed line shows the frequency-response expected for the D–A pair in the absence of D-to-A diffusion.

Figure 3.

Donor-to-acceptor distance distribution recovered from global analysis of the data obtained in propylene glycol at −20 and 20°C.

Figure 4.

Resolution of the recovered parameters (R_AV, HW and D) as seen from the ${\chi }_{\text{R}}^2$ surfaces. The solid lines are for global analysis of the data in propylene glycol at −20 and 20°C. The dotted lines (top) are for analysis of the data at a single temperature. The horizontal dashed line is the F-statistic for the degrees of freedom with a probability of P = 0.32.

Scheme 1.

Donor, acceptor and donor–acceptor pair (top to bottom) used to study slow end-to-end diffusion.