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Characterization of F-Actin Tryptophan Phosphorescence in the Presence and Absence of Tryptophan-Free Myosin Motor Domain

Emöke Bódis ^*, Giovanni B. Strambini , Margherita Gonnelli , András Málnási-Csizmadia  and Béla Somogyi ^*

^* Hungarian Academy of Sciences, Research Group for Fluorescence Spectroscopy, Office for Academy Research Groups Attached to Universities and Other Institutions, 7624 Pécs, Hungary; Consiglio Nazionale delle Ricerche, Istituto di Biofisica, 56100 Pisa, Italy; and Eötvös Loránd University, Department of Biochemistry, 1117 Budapest, Hungary

Correspondence: Address reprint requests to Prof. Béla Somogyi, Dept. of Biophysics, University of Pécs, Faculty of Medicine, Szigeti str. 12, H-7624 Pécs, Hungary. Tel.: 36-72-536260; Fax: 36-72-536261; E-mail: somogyi.publish{at}aok.pte.hu.

The effect of binding the Trp-free motor domain mutant of Dictyostelium discoideum, rabbit skeletal muscle myosin S1, and tropomyosin on the dynamics and conformation of actin filaments was characterized by an analysis of steady-state tryptophan phosphorescence spectra and phosphorescence decay kinetics over a temperature range of 140–293 K. The binding of the Trp-free motor domain mutant of D. discoideum to actin caused red shifts in the phosphorescence spectrum of two internal Trp residues of actin and affected the intrinsic lifetime of each emitter, decreasing by roughly twofold the short phosphorescence lifetime components (₁ and ₂) and increasing by 20% the longest component (₃). The alteration of actin phosphorescence by the motor protein suggests that i), structural changes occur deep down in the core of actin and that ii), subtle changes in conformation appear also on the surface but in regions distant from the motor domain binding site. When actin formed complexes with skeletal S1, an extra phosphorescence lifetime component appeared (₄, twice as long as ₃) in the phosphorescence decay that is absent in the isolated proteins. The lack of this extra component in the analogous actin-Trp-free motor domain mutant of D. discoideum complex suggests that it should be assigned to Trps in S1 that in the complex attain a more compact local structure. Our data indicated that the binding of tropomyosin to actin filaments had no effect on the structure or flexibility of actin observable by this technique.