Differential binding of tropomyosin isoforms to actin modified with m-maleimidobenzoyl-N-hydroxysuccinimide ester and fluorescein-5-isothiocyanate.
Differential interactions of tropomyosin (TM) isoforms with actin can be important for determination of the thin filament functions. A mechanism of tropomyosin binding to actin was studied by comparing interactions of five alphaTM isoforms with actin modified with m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) and with fluorescein-5-isothiocyanate (FITC). MBS attachment sites were revealed with mass spectrometry methods. We found that the predominant actin fraction was cross-linked by MBS within subdomain 3. A smaller fraction of the modified actin was cross-linked within subdomain 2 and between subdomains 2 and 1. Moreover, investigated actins carried single labels in subdomains 1, 2, and 3. Such extensive modification caused a large decrease in actin affinity for skeletal and smooth muscle tropomyosins, nonmuscle TM2, and chimeric TM1b9a. In contrast, binding of nonmuscle isoform TM5a was less affected. Isoform's affinity for actin modified in subdomain 2 by binding of FITC to Lys61 was intermediate between the affinity for native actin and MBS-modified actin except for TM5a, which bound to FITC-actin with similar affinity as to actin modified with MBS. The analysis of binding curves according to the McGhee-von Hippel model revealed that binding to an isolated site, as well as cooperativity of binding to a contiguous site, was affected by both actin modifications in a TM isoform-specific manner.
Skórzewski
Radosław
R
Institute of Experimental Biology, Kazimierz Wielki University in Bydgoszcz, 85-064 Bydgoszcz, Poland.
Robaszkiewicz
Katarzyna
K
Jarzebińska
Justyna
J
Suder
Piotr
P
Silberring
Jerzy
J
Moraczewska
Joanna
J
D000199:Actins / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D002853:Chromatography, Liquid; D004586:Electrophoresis; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry*; D013058:Mass Spectrometry; D008958:Models, Molecular; D036103:Nanotechnology; D011485:Protein Binding; D020033:Protein Isoforms / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D013194:Staining and Labeling; D013379:Substrate Specificity; D013388:Succinimides / Q000737:chemistry*; D014335:Tropomyosin / Q000378:metabolism*
D000199:Actins; D020033:Protein Isoforms; D013388:Succinimides; D014335:Tropomyosin; C013097:3-maleimidobenzoyl N-hydroxysuccinimide; D016650:Fluorescein-5-isothiocyanate