Proximity relationships and structural dynamics of the phalloidin binding site of actin filaments in solution and on single actin filaments on heavy meromyosin.
Distance relationships between phalloidin binding sites on F-actin have been investigated using fluorescence resonance energy transfer (FRET) techniques in solution and on single F-actin filaments bound to heavy meromyosin (HMM). Filaments saturated with an equimolar concentration of fluoresceinisothiocyanatophalloidin (FITC-ph) as the donor and tetramethylrhodamineisothiocyanatophalloidin (TRITC-ph) as the acceptor and control filaments saturated with either FITC-ph or TRITC-ph were characterized by absorption and fluorescence spectroscopy and the in vitro motility assay. Fluorescence excitation polarization spectroscopy showed hetero-FRET occurred within colabeled filaments, whereas homo-FRET was observed in control filaments. The distance measured between adjacent phalloidin binding sites using randomly labeled FITC-ph and TRITC-ph was 37.2 A using steady-state spectral analysis and 36.9 A using time-resolved spectroscopy with a radial coordinate of 14.5 A. Measurements of the distance between fluorescent phalloidin groups using the atomic model of F-actin [Lorenz, M., Popp, D., & Holmes, K. C. (1993) J. Mol. Biol. 234, 826-836] suggest transfer occurs between adjacent phalloidin molecules on opposite actin helices (39 A), rather than between adjacent phalloidins along the same actin helix (55.4 A). A quantitative fluorescence microscope technique was described that measures the proximity of adjacent FITC-ph and TRITC-ph on single filaments immobilized on HMM. Here a distance of 36.2 A was calculated which was unchanged during ATP-dependent sliding of F-actin on HMM. Spatially resolved FRET measurements are being used to observe the effects of actin binding proteins on the structural properties along the length of single actin filaments.
Heidecker
M
M
Biomolecular and Cellular Dynamics Research Group, Max Planck Institute for Biochemistry, Martinsried, Germany.
Yan-Marriott
Y
Y
Marriott
G
G
D000199:Actins / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002465:Cell Movement / Q000502:physiology; D004735:Energy Transfer; D066298:In Vitro Techniques; D008856:Microscopy, Fluorescence / Q000295:instrumentation; D008958:Models, Molecular; D015394:Molecular Structure; D015879:Myosin Subfragments / Q000737:chemistry* / Q000378:metabolism; D010590:Phalloidine / Q000031:analogs & derivatives / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011789:Quantum Theory; D011817:Rabbits; D012235:Rhodamines; D012996:Solutions; D013816:Thermodynamics
D000199:Actins; D015879:Myosin Subfragments; D012235:Rhodamines; D012996:Solutions; C037191:fluorescein isothiocyanate-phalloidin; D010590:Phalloidine; C041085:tetramethylrhodaminylphalloidine