Time-Resolved Energy Transfer and Conformational Distributions of Biopolymers

Abstract

In the previous chapter we described the principles of resonance energy transfer (RET), and how the phenomenon could be used to measure distances between donor and acceptor sites on macromolecules or the association between donor-labeled and acceptor-labeled biomolecules. Energy transfer was described as a through-space interaction that occurred whenever the emission spectrum of the donor overlapped with the absorption spectrum of the acceptor. For a given donor—acceptor (D—A) pair, the efficiency of energy transfer decreases as r −6, where r is the D—A distance. Each D—A pair has a characteristic distance: the Förster distance (R 0) at which RET is 50% efficient. The extent of energy transfer, as seen from the steady-state data, can be used to measure the distance, to study protein folding, to determine the extent of association based on proximity, or to create images of associated intracellular proteins. In this chapter we describe more advanced applications of RET, particularly those that rely on time-resolved measurements of covalently linked D—A pairs. For such pairs the time-resolved data can be used to recover the conformation-al distribution or distance distribution between the donor and acceptor. Donor-to-acceptor motions also influence the extent of energy transfer, which can be used to recover the mutual diffusion coefficient. In Chapter 15 we will consider RET between donors and acceptors that are not covalent-ly linked. In this case the extent of RET depends on the dimensional geometry of the molecules, such as planar distributions in membranes and one-dimensional distributions in double-helical DNA.