Structural Effects of Disease-Related Mutations in Actin-Binding Period 3 of Tropomyosin

Tropomyosin (Tpm) is an actin-binding coiled-coil protein. In muscle, it regulates contractions in a troponin/Ca<sup>2+</sup>-dependent manner and controls the thin filament lengths at the pointed end. Due to its size and periodic structure, it is difficult to observe small local structu...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Balaganesh Kuruba, Marta Kaczmarek, Małgorzata Kęsik-Brodacka, Magdalena Fojutowska, Małgorzata Śliwinska, Alla S. Kostyukova, Joanna Moraczewska
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	tropomyosin peptide stability congenital myopathy cardiomyopathy point mutations Organic chemistry QD241-441
Acceso en línea:	https://doaj.org/article/ffa9559956aa48ffac03a76114657829
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:ffa9559956aa48ffac03a76114657829
record_format	dspace
spelling	oai:doaj.org-article:ffa9559956aa48ffac03a761146578292021-11-25T18:28:53ZStructural Effects of Disease-Related Mutations in Actin-Binding Period 3 of Tropomyosin10.3390/molecules262269801420-3049https://doaj.org/article/ffa9559956aa48ffac03a761146578292021-11-01T00:00:00Zhttps://www.mdpi.com/1420-3049/26/22/6980https://doaj.org/toc/1420-3049Tropomyosin (Tpm) is an actin-binding coiled-coil protein. In muscle, it regulates contractions in a troponin/Ca<sup>2+</sup>-dependent manner and controls the thin filament lengths at the pointed end. Due to its size and periodic structure, it is difficult to observe small local structural changes in the coiled coil caused by disease-related mutations. In this study, we designed 97-residue peptides, Tpm1.1<sub>64–154</sub> and Tpm3.12<sub>65–155</sub>, focusing on the actin-binding period 3 of two muscle isoforms. Using these peptides, we evaluated the effects of cardiomyopathy mutations: I92T and V95A in Tpm1.1, and congenital myopathy mutations R91P and R91C in Tpm3.12. We introduced a cysteine at the N-terminus of each fragment to promote the formation of the coiled-coil structure by disulfide bonds. Dimerization of the designed peptides was confirmed by gel electrophoresis in the presence and absence of dithiothreitol. Using circular dichroism, we showed that all mutations decreased coiled coil stability, with Tpm3.12<sub>65–155</sub>R91P and Tpm1.1<sub>64–154</sub>I92T having the most drastic effects. Our experiments also indicated that adding the N-terminal cysteine increased coiled coil stability demonstrating that our design can serve as an effective tool in studying the coiled-coil fragments of various proteins.Balaganesh KurubaMarta KaczmarekMałgorzata Kęsik-BrodackaMagdalena FojutowskaMałgorzata ŚliwinskaAlla S. KostyukovaJoanna MoraczewskaMDPI AGarticletropomyosinpeptidestabilitycongenital myopathycardiomyopathypoint mutationsOrganic chemistryQD241-441ENMolecules, Vol 26, Iss 6980, p 6980 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	tropomyosin peptide stability congenital myopathy cardiomyopathy point mutations Organic chemistry QD241-441
spellingShingle	tropomyosin peptide stability congenital myopathy cardiomyopathy point mutations Organic chemistry QD241-441 Balaganesh Kuruba Marta Kaczmarek Małgorzata Kęsik-Brodacka Magdalena Fojutowska Małgorzata Śliwinska Alla S. Kostyukova Joanna Moraczewska Structural Effects of Disease-Related Mutations in Actin-Binding Period 3 of Tropomyosin
description	Tropomyosin (Tpm) is an actin-binding coiled-coil protein. In muscle, it regulates contractions in a troponin/Ca<sup>2+</sup>-dependent manner and controls the thin filament lengths at the pointed end. Due to its size and periodic structure, it is difficult to observe small local structural changes in the coiled coil caused by disease-related mutations. In this study, we designed 97-residue peptides, Tpm1.1<sub>64–154</sub> and Tpm3.12<sub>65–155</sub>, focusing on the actin-binding period 3 of two muscle isoforms. Using these peptides, we evaluated the effects of cardiomyopathy mutations: I92T and V95A in Tpm1.1, and congenital myopathy mutations R91P and R91C in Tpm3.12. We introduced a cysteine at the N-terminus of each fragment to promote the formation of the coiled-coil structure by disulfide bonds. Dimerization of the designed peptides was confirmed by gel electrophoresis in the presence and absence of dithiothreitol. Using circular dichroism, we showed that all mutations decreased coiled coil stability, with Tpm3.12<sub>65–155</sub>R91P and Tpm1.1<sub>64–154</sub>I92T having the most drastic effects. Our experiments also indicated that adding the N-terminal cysteine increased coiled coil stability demonstrating that our design can serve as an effective tool in studying the coiled-coil fragments of various proteins.
format	article
author	Balaganesh Kuruba Marta Kaczmarek Małgorzata Kęsik-Brodacka Magdalena Fojutowska Małgorzata Śliwinska Alla S. Kostyukova Joanna Moraczewska
author_facet	Balaganesh Kuruba Marta Kaczmarek Małgorzata Kęsik-Brodacka Magdalena Fojutowska Małgorzata Śliwinska Alla S. Kostyukova Joanna Moraczewska
author_sort	Balaganesh Kuruba
title	Structural Effects of Disease-Related Mutations in Actin-Binding Period 3 of Tropomyosin
title_short	Structural Effects of Disease-Related Mutations in Actin-Binding Period 3 of Tropomyosin
title_full	Structural Effects of Disease-Related Mutations in Actin-Binding Period 3 of Tropomyosin
title_fullStr	Structural Effects of Disease-Related Mutations in Actin-Binding Period 3 of Tropomyosin
title_full_unstemmed	Structural Effects of Disease-Related Mutations in Actin-Binding Period 3 of Tropomyosin
title_sort	structural effects of disease-related mutations in actin-binding period 3 of tropomyosin
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/ffa9559956aa48ffac03a76114657829
work_keys_str_mv	AT balaganeshkuruba structuraleffectsofdiseaserelatedmutationsinactinbindingperiod3oftropomyosin AT martakaczmarek structuraleffectsofdiseaserelatedmutationsinactinbindingperiod3oftropomyosin AT małgorzatakesikbrodacka structuraleffectsofdiseaserelatedmutationsinactinbindingperiod3oftropomyosin AT magdalenafojutowska structuraleffectsofdiseaserelatedmutationsinactinbindingperiod3oftropomyosin AT małgorzatasliwinska structuraleffectsofdiseaserelatedmutationsinactinbindingperiod3oftropomyosin AT allaskostyukova structuraleffectsofdiseaserelatedmutationsinactinbindingperiod3oftropomyosin AT joannamoraczewska structuraleffectsofdiseaserelatedmutationsinactinbindingperiod3oftropomyosin
_version_	1718411114688020480

Structural Effects of Disease-Related Mutations in Actin-Binding Period 3 of Tropomyosin

Ejemplares similares