The measurement of the efficiency of Förster long-range resonance energy transfer between donor (D) and acceptor (A) luminophores attached to the same macromolecular substrate can be used to estimate the D-A separation, R. If the D and A transition dipoles sample all orientations with respect to the substrate (the isotropic condition) in a time short compared with the transfer time (the dynamic averaging condition), the average orientation factor less than K2 greater than is 2/3. If the isotropic condition is not satisfied but the dynamic averaging condition is, upper and lower bounds for less than K2 greater than, and thus R, may be obtained from observed D and A depolarizations, and these limits may be further narrowed if the transfer depolarization is also known. This paper offers experimental protocols for obtaining this reorientational information and presents contour plots of less than K2 greater than min and less than K2 greater than max as functions of generally observable depolarizations. This permits an uncertainty to be assigned to the determined value of R. The details of the D and A reoreintational process need not be known, but the orientational distributions are assumed to have at least approximate axial symmetry with respect to a stationary substrate. Average depolarization factors are derived for various orientational distribution functions that demonstrate the effects of various mechanisms for reorientation of the luminophores. It is shown that in general the static averaging regime does not lend itself to determinations of R.

A new method is described for measuring motions of protein domains in their native environment on the physiological timescale. Pairs of cysteines are introduced into the domain at sites chosen from its static structure and are crosslinked by a bifunctional rhodamine. Domain orientation in a reconstituted macromolecular complex is determined by combining fluorescence polarization data from a small number of such labelled cysteine pairs. This approach bridges the gap between in vitro studies of protein structure and cellular studies of protein function and is used here to measure the tilt and twist of the myosin light-chain domain with respect to actin filaments in single muscle cells. The results reveal the structural basis for the lever-arm action of the light-chain domain of the myosin motor during force generation in muscle.

SynopsisThe dependence of Forster long-range resonance energy transfer efficiency on the orientational freedom of donor D and acceptor A molecules attached to a macromolecular substrate is examined. The usefulness of polarized emission measurements in determining the mutual orientation as well as the degree of orientational freedom of D and A and thereby deriving maximum and minimum values for the D-A separation from the transfer efficiency is demonstrated. INTRODUCTlONAn exact theory of resonance transfer of electronic excitation energy between isolated donor D and acceptor A molecules of suitable spectroscopic properties was first presented almost a quarter of a century ago by Forster.'V2 The theory was immediately successfully applied to the phenomenon of quenching of donor fluorescence in mixed solutions of donor and acceptor dyestuffs3 and to that of concentration depolarization of viscous solutions of a single d y e~t u f f ,~ and has enjoyed continued success and widespread use in these5s6 and somewhat more s p e~i a l i z e d~-~~ systems. However, it was not until considerably later that the predicted dependence of transfer rate on the inverse sixth power of the intramolecular D-A separation R was demonstrated, in several laboratories, for isolated D-A pairs covalently attached to a neutral substrate molecule. [11][12][13][14] Only then did the practicability of using the energy transfer method as a "spectroscopic ruler"12 for the determination of intramolecular separations of the order of the dimensions of macromolecules (up to several tens of angstroms), which are of interest to physical organic chemists and more especially polymer chemists and biochemists, become widely recognized. Thus, the last few years have seen an increasing literature on the application of this technique to the determination of the topology of, in particular, biopolym e r~. l~-~* In almost all this work a major difficulty in interpretation has presented itself in that the mutual orientation of the D-A pair, which could * Present address:

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A fluorescent phospholipid derivative, the fluoresceinthiocarbamyl adduct of a natural phosphatidylethanolamine, has been synthesized and incorporated into sonicated single-bilayer vesicles of egg lecithin and dipalmitoyllecithin. The surface location of this probe has been confirmed by using extrinsic fluorescence quenching studies together with steady-state emission anisotropy measurements. Electronic excitation energy transfer between 1,6-diphenyl-1,3,5-hexatriene incorporated within the hydrophobic core of the bilayer and the novel derivative has been investigated to estimate the depth within the bilayer at which the former is located. Efficiencies have been measured for two different phospholipids, egg lecithin and dipalmitoyllecithin, in the latter case both above and below the phospholipid phase transition, with and without added cholesterol. The observed dependence of the transfer efficiency on the acceptor concentration was compared with that calculated according to Förster theory applied to random two-dimensional distributions of donor and acceptor molecules in parallel planes for various interplanar separations, taking into account orientational effects. The Förster R0 of about 45 A for this donor-acceptor pair is particularly well suited to such studies since it is of the order of the width of the bilayer. The experiments showed that energy-transfer spectroscopy can provide useful quantitative information as to the transverse location of diphenylhexatriene in homogeneous phospholipid bilayers and may also reflect lateral partitioning of donor or of both donor and acceptor into different phases in systems exhibiting phase separations.

The orientation of proteins in ordered biological samples can be investigated using steady-state polarized fluorescence from probes conjugated to the protein. A general limitation of this approach is that the probes typically exhibit rapid orientational motion ("wobble") with respect to the protein backbone. Here we present a method for characterizing the extent of this wobble and for removing its effects from the available information about the static orientational distribution of the probes. The analysis depends on four assumptions: 1) the probe wobble is fast compared with the nanosecond time scale of its excited-state decay; 2) the orientational distributions of the absorption and emission transition dipole moments are cylindrically symmetrical about a common axis c fixed in the protein; 3) protein motions are negligible during the excited-state decay; 4) the distribution of c is cylindrically symmetrical about the director of the experimental sample. In a muscle fiber, the director is the fiber axis, F. All of the information on the orientational order of the probe that is available from measurements of linearly polarized fluorescence is contained in five independent polarized fluorescence intensities measured with excitation and emission polarizers parallel or perpendicular to F and with the propagation axis of the detected fluorescence parallel or perpendicular to that of the excitation. The analysis then yields the average second-rank and fourth-rank order parameters ( and ) of the angular distribution of c relative to F, and and , the average second-rank order parameters of the angular distribution for wobble of the absorption and emission transition dipole moments relative to c. The method can also be applied to other cylindrically ordered systems such as oriented lipid bilayer membranes and to processes slower than fluorescence that may be observed using longer-lived optically excited states.

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