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Differential Binding of Tropomyosin Isoforms to Actin Modified With m-maleimidobenzoyl-N-hydroxysuccinimide Ester and fluorescein-5-isothiocyanate

Differential Binding of Tropomyosin Isoforms to Actin Modified With m-maleimidobenzoyl-N-hydroxysuccinimide Ester and fluorescein-5-isothiocyanate

Radosław Skórzewski, Katarzyna Robaszkiewicz, Justyna Jarzebińska, Piotr Suder, Jerzy Silberring, Joanna Moraczewska

Anal Biochem. 2009 Nov 1;394(1):48-55.

PMID: 19583962

Abstract:

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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
IAR42418203	Fluorescein-5-EX N-hydroxysuccinimide ester			Price

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