Use of Nonspecific Dye Labeling for Singlet Energy-Transfer Measurements in Complex Systems. A Simple Model

Techniques are described which will permit the semiquantitative interpretation of singlet-singlet energy-transfer measurements on multiply labeled single proteins and protein complexes. The two critical assumptions are that fluorescent chromophores can be placed anywhere at random on a protein surface and that the stoichiometry of labeling is governed by a Poisson distribution. In calculations on protein complexes it is further assumed that one can exclusively localize donors on the surface of one protein, and acceptors on another. Calculations were initially carried out to allow for the occurrence of donor-donor energy transfer. These show that in most cases of interest one can neglect this process. Singlet energy-transfer measurements on a double-labeled protein will permit measurement of the anhydrous radius. The results are quite insensitive to variations in axial ratio if the volume is kept constant. In a complex of two or more proteins, singlet energy-transfer measurements will enable fairly accurate determination of the distances between pairs of proteins. For most accurate results in both cases one should attempt to work at ratios of several acceptor molecules per protein. This minimizes errors due to uncertainties in the extinction of bound dyes. The average number of donor molecules per protein is not important. Examples of the application of these calculations are given in the accompanying paper.