Fluorescent Probes Attached to Cys 35 or Cys 84 in Cardiac Troponin C Are Differentially Sensitive to Ca2+-Dependent Events in Vitro and in Situ

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• 作者：John A. Putkey ; Wen Liu ; Xin Lin ; Saadia Ahmed ; Mei Zhang ; James D. Potter ; and W. Glenn L. Kerrick
• 刊名：Biochemistry
• 出版年：1997
• 出版时间：January 28, 1997
• 年：1997
• 卷：36
• 期：4
• 页码：970 - 978
• 全文大小：236K
• 年卷期：v.36,no.4(January 28, 1997)
• ISSN：1520-4995

文摘

The goal of the current study was to generate recombinant cTnCproteins with single Cysresidues as sites for attachment of fluorescent probes that candistinguish between the structural effectsof myosin cross bridges and direct Ca²⁺ binding to cTnC(cardiac and slow skeletal troponin C) in skinnedfibers. We anticipated that cTnC proteins which retain theendogenous Cys 35 (cTnC(C35)) or Cys 84(cTnC(C84)) would provide fluorescent probes with distinctmicroenvironments, since these residues areon opposite sides of the globular regulatory domain. Invitro experiments that showed IAANS (2-(4'-(iodoacetamido)anilino)naphthalene-6-sulfonic acid) coupled toCys 35 can induce unwanted structuralperturbations as evidenced by a decreased affinity of site II forCa²⁺ when IAANS-labeled cTnC(C35) isbound to cTnI. Important structural features involving Cys 35 inthe inactive site I are suggested by aCa²⁺-dependent increase in reactivity of Cys 35 withsulfhydryl specific reagents when cTnC(C35) isassociated with cTnI. These characteristics are not seen forcTnC(C84). When incorporated in situintoskinned cardiac muscle fibers, native cTnC with IAANS bound to both Cys35 and Cys 84 showed apCa₅₀ of fluorescence which preceded that of force, whilethe pCa₅₀ values of both force andfluorescencewere coincident for IAANS-labeled cTnC(C84). Disruption offorce-producing myosin cross bridgeshad no effect on the pCa₅₀ of fluorescence forIAANS-labeled cTnC(C84), but induced a rightwardshiftin the pCa₅₀ of fluorescence for IAANS-labeled native cTnC.These data can be interpreted to indicatethat cTnC with IAANS bound to both Cys 35 and C84 senses either myosincross bridges or direct Ca²⁺binding and myosin-induced cooperativity, while IAANS bound to Cys 84alone senses conformationsthat are tightly coupled with force generation.