Single-Molecule Conformational Dynamics Probed by Electron Transfer

The conformational dynamics of a biomolecule is crucial to its biological functions, but these motions are usually spontaneous and unsynchronized and subsequently difficult to probe in ensemble-averaged experiments. The single molecule technique is suitable for conformational dynamics study as it avoids the need of synchronization.

Fluorescence Quenching in NADH:flavin Oxidoreductase (Fre)

In order to study protein dynamics without perturbation, we utilize a naturally fluorescent flavin enzyme Fre using the fluorescent isoalloxazine of flavins as a probe. On the left is the structure of a Fre/FAD complex. In contrast to the free isoalloxazine fluorescence with a mono-exponential decay with a long lifetime, the decay of Fre-bound flavins is much faster and is multi-exponential. The fast fluorescence decay has been attributed to quenching of the excited state by ET from nearby residues tyrosines. There are three tyrosines around FAD. Through site-directed mutagenesis, our collaborator Luying Xun at Washing State University proved that tyrosine35 is determined to be the major quencher. The figure in the right are lifetime decays of the wt and mutant Fre/FMN complexes. Due to the higher binding affinity of FAD to Fre than over FMN, we choose FAD/Fre complex for single molecule study.

Probing Conformational Dynamics by Excited State Electron Transfer

We use excited state electron transfer (ET) to probe the subtle structure change within biomolecules. The following is the energy diagram of the excited state ET.

The fluorescence lifetime t is determined by the radiative rate g and the non-radiative relaxation rates. In the case when the ET dominates the relaxation rate, the fluorescence lifetime t = 1/( g + kET ). The ET rate is highly dependent on the edge to edge distance between the electron donor and acceptor following kET=k0*exp(- b R) . Therefore the fluctuation of the donor-acceptor distance R results in the fluctuation of the fluorescence lifetime t . The scaling factor b is around 1.0~1.4 Å -1 for proteins, which makes ET a distance dependent probe for the subtle motions on angstrom scale. The amino acid residues tyrosine and tryptophan or DNA base Guanine can serve as natural ET donors. A typical ET chromophore can be natural chromophores such as flavin, or artificial dyes such as Rhodamines. Single molecule ET could be applied to study the conformational dynamics, complementary to the widely adopted Fluorescence Resonance Energy Transfer (FRET) technique, which is sensitive for motion on nanometer scale.

Probing Single Molecule Dynamics Photon-by-Photon

We use a confocal microscope to detect the fluorescence signal from a single molecule. Different from the conventional time correlated single photon counting (TCSPC), for each detected fluorescence photon with index, p, we record both the delay time respect to its excitation pulse, t p , and the chronological arrival time tp (see the accompany figure). Instead of binning the detected photon to calculate the fluorescence lifetime, the lifetime correlation function C(t) can be calculated with the novel photon-by-photon correlation method. Such a method provides dynamic information with a high time resolution and a broad range of time scale.

Conformational Dynamics of Fre

We probe the distance fluctuations between FAD and tyr35 in real time through the flavin (indicated by the light bulb) fluorescence lifetime. The time trace of fluorescence lifetime reflects the distance fluctuation in the single enzyme molecule, which takes place on a very broad time scale range.

The scheme on the right ( see enlarged version ) shows the apparatus for single molecule electron transfer measurement, combining an inverted fluorescence microscope, mode-locked Ti:sapphire laser, time correlated photon counting electronics, a single-photon timing module (SPTM) containing a fast single-photon avalanche diode (SPAD) developed by Cova et al. that provides a fast time response (with 60ps FWHM).

The fluorescence decay of a single wt-Fre/FAD complex immobilized on the quartz surface is multi-exponential (left figure), suggesting fluctuations of fluorescence lifetime during the course of measurement. The lifetime correlation function shows the fluctuation occurrs at a broad range of time scales, from hundreds of microseconds to tens of seconds.

The correlation function can be fitted with a stretched-exponential exp[-(t/ t 0)^ b ] with t 0 = 54 ms and b=0.30 (right figure).

The coarse-grained lifetime distribution can be extracted from the single-molecule lifetime trajectory. From the lifetime distribution, the potential of mean force of the distance between the tyrosine35 and isoalloxazine chromophore is obtained and can be modeled with a parabolic potential (left figure).

To understand the conformational dynamics, we model the system with a fictitious particle undergoing diffusional motion within the potential mean force. However the Brownian diffusion theory predicts a single exponential correlation function. In collaboration with Sam Kou of Havard statistics, we have developed a model of subdiffusion within a rugged energy landscaped based on the generalized Langevin equation. This model can account for the experimental findings as well.

The highly stretched lifetime correlation obtained here implies a broad range of traping times, corresponding to broadly distributed energy barriers between different conformers. The existence of multiple meta-stable conformers is related to the fluctuating enzymatic activity now seen on several systems. (Lu et al. Science 282, 1877 (1998), van Oijen et al. Science 301, 1235 (2003)).

Publications

1. H. Yang; X. S. Xie, Chem. Phys, 284, 423(2002).
2. T. Louie, H. Yang, P. Karnchanaphanurach, X. S. Xie, L.-Y. Xun, J. Biol. Chem. 277, 39450 (2002).
3 X. S. Xie, J. Chem. Phys., 117, 11024(2002).
4. H. Yang, X. S. Xie, J. Chem. Phys. 117, 10965(2002).
5 . A. M. van Oijen, P. C. Blainey, J. D. Crampton, C. C. Richardson, T. Ellenberger and X. S. Xie, Science, 301, 1235(2003 ).
6. H. Yang, G. Luo, P. Karnchanaphanurach, T. Louie, L. Xun, I. Rech, S. Cova and X. S. Xie, Science 302, 262(2003).