ARTICLE IN PRESS Two opposite effects of cofilin on the thermal unfolding of F-actin: a differential scanning calorimetric study Irina V. Dedova a , Olga P. Nikolaeva b , Valeria V. Mikhailova c , Cris G. dos Remedios a , Dmitrii I. Levitsky b,c, * a Muscle Research Unit, Department of Anatomy and Histology, The University of Sydney, NSW 2006 Sydney, Australia b Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119992, Russia c Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky prosp. 33, Moscow 119071, Russia Received 11 October 2003; received in revised form 24 January 2004; accepted 27 January 2004 Abstract Differential scanning calorimetry was used to examine the effects of cofilin on the thermal unfolding of actin. Stoichiometric binding increases the thermal stability of both G- and F-actin but at sub-saturating concentrations cofilin destabilizes F-actin. At actin:cofilin molar ratios of 1.5 – 6 the peaks corresponding to stabilized (66 – 67 jC) and destabilized (56 – 57 jC) F-actin are observed simultaneously in the same thermogram. Destabilizing effects of sub-saturating cofilin are highly cooperative and are observed at actin:cofilin molar ratios as low as 100:1. These effects are abolished by the addition of phalloidin or aluminum fluoride. Conversely, at saturating concentrations, cofilin prevents the stabilizing effects of phalloidin and aluminum fluoride on the F-actin thermal unfolding. These results suggest that cofilin stabilizes those actin subunits to which it directly binds, but destabilizes F-actin with a high cooperativity in neighboring cofilin-free regions. D 2004 Elsevier B.V. All rights reserved. Keywords: Cofilin; Actin; Thermal unfolding; Differential scanning calorimetry 1. Introduction Actin filaments and their dynamics play essential roles in the development and function of eukaryotic organisms. A large number of actin-binding proteins can control assembly, disassembly, and rearrange- ment of actin filaments [1]. Small proteins that belong to the actin-depolymerizing factor (ADF)/ cofilin family play a central role in the regulation of actin dynamics in cells. These proteins dramat- ically alter actin microfilament assembly, probably by accelerating the off-rate for actin subunits from the pointed ends of the filaments [2–4]. Cofilin can also depolymerize and sever actin filaments [5]. Cofilin binds stoichiometrically to both monomeric (G-) and polymeric (F-) actin [6]. Binding of cofilin alters the conformation of the so-called small do- main of actin, reducing the distance between probes attached to specific sites in subdomains 1 and 2 [7]. A model proposed for the decorated filaments [8] suggests that at least one binding site for cofilin lies between successive monomers along the long- pitch, making contacts with the lower subdomains 0301-4622/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bpc.2004.01.009 Abbreviations: ADF, actin depolymerizing factor; DSC, differ- ential scanning calorimetry.. * Corresponding author. Fax: +7-095-954-2732 E-mail address: levitsky@inbi.ras.ru (D.I. Levitsky). www.elsevier.com/locate/bpc BIOCHE-04375; No. of pages: 10 Biophysical Chemistry 110 (2004) 119– 128