Determination of fluorescent probe orientations on biomolecules by conformational searching: Algorithm testing and applications to the atomic model of myosin

The ability of a localized conformational searching method to predict probe orientation was tested on model nucleic acid and protein structures and ap plied to the prediction of skeletal myosin integrity upon chemical modifica tion of its reactive thiols. Double-stranded oligonucleotides were chemical ly labeled with donor and acceptor resonance energy transfer probes at each end for distance determinations. These measurements were made independentl y using a terbium chelate as a donor to each of four chemically and spectro scopically distinct acceptor probes from the xanthene and cyanine dye group s. The choice of acceptor significantly affected the separation distance me asured. Conformational searching algorithms on the atomic model corrected f or the differences to within 0.2 nm on average. Verifying its usefulness on proteins, the localized conformational searching method determined the ori entation of a fluorescent probe on RNase A that corresponds closely to avai lable crystallographic models of the labeled protein (RMS deviation = 0.1 n m). Also, analysis of the symmetry of the fluorophores' structures suggests why FRET orientation factors are often closer to their dynamic average val ue than might normally be expected. Furthermore, the computational method p rovides insights about FRET data that are important for assessing the stabi lity of the a helix separating the SH1 and SH2 reactive thiols in skeletal myosin. (C) 1999 Academic Press.