Cross-Linking between the Regulatory Regions of Troponin-I and Troponin-C Abolishes the Inhibitory Function of Troponin

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• 作者：Yin Luo ; Bing Li ; Guang Yang ; John Gergely ; and Terence Tao
• 刊名：Biochemistry
• 出版年：2002
• 出版时间：October 22, 2002
• 年：2002
• 卷：41
• 期：42
• 页码：12891 - 12898
• 全文大小：374K
• 年卷期：v.41,no.42(October 22, 2002)
• ISSN：1520-4995

文摘

We reported previously that both residues 48 and 82 on opposite sides of troponin-C's (TnC's)N-terminal regulatory hydrophobic cleft photo-cross-linked to Met121 of troponin-I (TnI) [Luo, Y., Leszyk,J., Qian, Y., Gergely, J., and Tao, T. (1999) Biochemistry 38, 6678-6688]. Here we report that the Ca²⁺-absent inhibitory activity of troponin (Tn) was progressively lost as the extent of photo-cross-linkingincreased. To extend these studies, we constructed a mutant TnI with a single cysteine at residue 121(TnI121). In Tn complexes containing TnI121 and mutant TnCs with a single cysteine at positions 12,48, 82, 98, or 125 (TnC12, TnC48 etc.), TnI121 formed disulfide cross-links primarily with TnC48 andTnC82 when Ca²⁺ was present, and with only TnC48 when Ca²⁺ was absent. These results indicate thatTnI Met121 is situated within the N-domain hydrophobic cleft of TnC in the presence of Ca²⁺, and thatit moves out of the cleft upon Ca²⁺ removal but remains within the vicinity of TnC. Activity assaysrevealed that the Met121 to Cys mutation in TnI121 reduced the Ca²⁺-present activation of Tn, indicatingthat Met121 is important in hydrophobic interactions between this TnI region and TnC's N-domain cleft.The formation of a disulfide cross-link between TnI121 and TnC48 or TnC82 abolished the Ca²⁺-absentinhibitory activity of Tn, indicating that the movement of the Met121 region of TnI out of TnC's N-domaincleft is essential for the occurrence of further events in the inhibitory process of skeletal muscle contraction.On the basis of these and other results, a simple mechanism for Ca²⁺ regulation of skeletal musclecontraction is presented and discussed.