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Type:	Artigo de Periódico
Title:	Weak domain stability and higher ca 2+ binding affinity contribute to allostery between the d/e linker and n-helix of cardiac troponin c
Authors:	Mayra de Amorim Marques Adolfo Henrique de Moraes Silva Jerson Lima da Silva Jose R. Pinto Guilherme Augusto Piedade de Oliveira
Abstract:	Hypertrophic cardiomyopathy (HCM) is an inherited myopathy caused by the production of anomalous sarcomeric proteins that can lead to severe cardiac dysfunction. Here, we used structural and biophysical approaches to better understand the pathogenesis of a cardiac troponin C (cTnC) C84Y mutation located in the D/E linker, first reported in a 17-year-old proband, presenting with left-ventricular hypertrophy. Despite the relevance of HCM disease, little is known concerning the function of the D/E linker and allosteric phenomena governing cTnC Ca2+ affinity. Monitored by bis-ANS fluorescence, Ca2+-titrations reveal that C84Y exhibits enhanced Ca2+-binding affinity in both domains and conformational changes compared to WT. Although WT and C84Y display distinct Ca2+-binding behaviors, the overall dimensional values and molecular envelopes generated by small-angle-X-ray scattering data remains similar. Using circular-dichroism, C84Y revealed significantly lower thermostability in non-Ca2+-bound form compared to WT. Most of our understanding of the molecular mechanisms underlying how troponin and troponin peptides switch muscle contraction “on” and “off” has been derived using experimental NMR techniques. Currently, no experimental techniques are available that allow the understanding of protein regulatory/dynamic processes at the molecular level of large, multi-domain protein complexes. To further unravel molecular changes in C84Y, three-dimensional NMR experiments were performed for backbone assignment. The largest chemical shifts were observed in N-Helix residues and at the end of D-helix and D/E linker. NMR-derived backbone amide temperature-coefficients indicate different temperature-dependent conformational changes exist between WT and C84Y Carr-Purcell-Meiboom-Gill relaxation dispersion (CPMG-RD) and R1/R2 experiments were used to probe the population and exchanging rates of C84Y compared to WT. This work sought to elucidate: main structural components underlying this pathological mutation, novel allosteric mechanisms, and the role of D/E linker in cTnC.
Subject:	Batimento cardíaco Enzimas alostéricas Miocárdio Doenças Mutação (Biologia)
language:	eng
metadata.dc.publisher.country:	Estados unidos
Publisher:	Universidade Federal de Minas Gerais
Publisher Initials:	UFMG
metadata.dc.publisher.department:	ICX - DEPARTAMENTO DE QUÍMICA
Rights:	Acesso Aberto
metadata.dc.identifier.doi:	https://doi.org/10.1016/j.bpj.2017.11.2336
URI:	http://hdl.handle.net/1843/46067
Issue Date:	2-Feb-2018
metadata.dc.url.externa:	https://www.sciencedirect.com/science/article/pii/S0006349517335683?via%3Dihub
metadata.dc.relation.ispartof:	Biophysical Journal
Appears in Collections:	Artigo de Periódico

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