Biophys J, November 2002, p. 2726-2732, Vol. 83, No. 5

Thin Filament Regulation and Ionic Interactions between the N-terminal Region in Actin and Troponin

Wenise W. Wong,^* Jack H. Gerson,^* Peter A. Rubenstein, and Emil Reisler^*

 ^*Department of Chemistry and Biochemistry and the Molecular Biology Institute, University of California, Los Angeles, California 90095 and  Department of Biochemistry, College of Medicine, University of Iowa, Iowa City, Iowa 52242 USA

The N-terminal region in actin has been shown to interact with both myosin and troponin (Tn) during the cross-bridge cycle and in regulation. To study the role of this region in regulation, we used yeast actin mutants with increased and decreased numbers of acidic residues. The mutants included D24A/D25A, with Asp²⁴ and Asp²⁵ replaced with alanines; DNEQ, with the substitution of Asp² and Glu⁴ with their amide analogs; and 4Ac, with Glu³ and Asp⁴ inserted in lieu of Ser³. In the in vitro motility assay, using reconstituted regulated thin filaments, the sliding speeds of DNEQ, D24A/D25A, and 4Ac were similar at all pCa values. Thus, Ca²⁺-sensitivity of the thin filaments and the inhibitory function of TnI appear to be insensitive to changes in charge (±2) at the N-terminus of actin, suggesting little, if any, role of that actin region in regulation. A Ca²⁺-independent conformational change in that region was detected upon troponin binding to actin-Tm via an increase in the fluorescence of a pyrene probe attached to another yeast actin mutant that we used (Cys¹).

Biophys J, November 2002, p. 2726-2732, Vol. 83, No. 5
© 2002 by the Biophysical Society   0006-3495/02/11/2726/07  $2.00