Construção e análise de mutantes fluorescentes da troponina I / Construction and analysis of fluorescent mutants of troponin I

AUTOR(ES)

Deodoro Camargo Silva Gonçalves de Oliveira

DATA DE PUBLICAÇÃO

2001

RESUMO

Vertebrate striated muscle contraction is regulated by troponin (Tn). Tn is composed of three subunits: troponin I (TnI), troponin C (TnC) and troponin T (TnT). TnI has an inhibitory role that is neutralized by calcium binding to the regulatory sites in the N-domain of TnC, and TnT positions the troponin complex on the thin filament. In order to follow the Ca2+ induced conformational change that is transmitted from TnC to TnI, the unique spectral properties of 5-hydroxytryptophan (5HW) incorporated as point-mutants of TnI were used. It was possible to identify two new TnI intrinsic spectral probes sensitive to Ca2+ binding to Tn: TnI with single 5HW at positions 100 and 121. Trimeric troponin complexes reconstituted with two fluorescent mutants of TnI, Tn-TnIF100HW and Tn-TnIM121HW, showed respectively 12 and 70 % increase in the emission spectra when Ca2+ bound to TnC. In the binary complexes (TnC-TnI) two TnIs with 5HW at positions 106 and 121 were also sensitive to Ca2+ binding to TnC. Fluorescence analysis of these probes showed: 1) the regions in TnI that respond to Ca2+ binding to the regulatory N-domain of TnC are the inhibitory region of TnI (residues 96 to 116), and a neighbor region that includes position 121; 2) point mutations and incorporation of 5HW in TnI can affect both the affinity and the cooperativity of Ca2+ binding to TnC, confirming the role of TnI as a modulator of the Ca2+ affinity of TnC; 3) the high dissociation constant for sites in the N-terminal domain of TnC (Kd ~ 10-8 M), derived from data using probes in the inhibitory region of TnI suggested the possibility that these sites are the high affinity Ca2+ binding sites in the troponin complex.

ASSUNTO(S)

estrutura e função de proteínas fuorescência troponina fluorescence site-directed mutagenesis mutação sítio-dirigida protein structure and function regulação da contração muscular troponin regulation of muscle contraction

Documentos Relacionados

• Expressão de troponina I do musculo esqueletico de galinha em E. coli
• Estudo da atividade inibitória da troponina I através de mutações sítio-dirigidas
• Construction and evaluation of microsystems for flow analysis
• Construção e caracterização de linhagens de Salmonella enterica mutantes nos genes de IHF (Integral Host Factor)
• Construction and analysis of a photometric elipsometer with rotating compensator.