Proximity relationships and structural dynamics of the phalloidin binding site of actin filaments in solution and on single actin filaments on heavy meromyosin

• Published in: Biochemistry (Easton) Vol. 34; no. 35; pp. 11017 - 11025
• Main Authors: Heidecker, Manfred, Yan-Marriott, Yuling, Marriott, Gerard
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 05.09.1995
• Subjects: Actins - chemistry; Actins - metabolism; Animals; Binding Sites; Cell Movement - physiology; Energy Transfer; In Vitro Techniques; Microscopy, Fluorescence - instrumentation; Models, Molecular; Molecular Structure; Myosin Subfragments - chemistry; Myosin Subfragments - metabolism; Phalloidine - analogs & derivatives; Phalloidine - metabolism; Protein Binding; Protein Conformation; Quantum Theory; Rabbits; Rhodamines; Solutions; Thermodynamics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00035a007

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.