Thin filament regulation and ionic interactions between the N-terminal region in actin and troponin.
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 Asp(24) and Asp(25) replaced with alanines; DNEQ, with the substitution of Asp(2) and Glu(4) with their amide analogs; and 4Ac, with Glu(3) and Asp(4) inserted in lieu of Ser(3). 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, Ca(2+)-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 Ca(2+)-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 (Cys(1)).
Wong
Wenise W
WW
Department of Chemistry and Biochemistry, Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA.
Gerson
Jack H
JH
Rubenstein
Peter A
PA
Reisler
Emil
E
D000199:Actins / Q000737:chemistry* / Q000378:metabolism; D000409:Alanine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D002118:Calcium / Q000737:chemistry; D005456:Fluorescent Dyes / Q000494:pharmacology; D007477:Ions; D008958:Models, Molecular; D009068:Movement; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011721:Pyrenes / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014336:Troponin / Q000737:chemistry* / Q000378:metabolism
D000199:Actins; D005456:Fluorescent Dyes; D007477:Ions; D011721:Pyrenes; D014336:Troponin; D001224:Aspartic Acid; D000409:Alanine; D002118:Calcium