Kobayashi T, Kobayashi M, Gryczynski Z, Lakowicz J R, Collins J H
Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore 21201, USA.
Biochemistry. 2000 Jan 11;39(1):86-91. doi: 10.1021/bi991903b.
In muscle thin filaments, the inhibitory region (residues 96-117) of troponin I (TnI) is thought to interact with troponin C (TnC) in the presence of Ca(2+) and with actin in the absence of Ca(2+). To better understand these interactions, we prepared mutant TnIs which contained a single Cys-96 or Cys-117 and labeled them with the thiol-specific fluorescent probe N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine (IAEDANS). We characterized the microenvironments of the AEDANS labels on TnI in the presence and absence of Ca(2+) by measuring the extent of acrylamide quenching of fluorescence and lifetime-resolved anisotropy. In the troponin-tropomyosin (Tn-Tm) complex, the AEDANS labels on both Cys-96 and Cys-117 were less accessible to solvent and less flexible in the presence of Ca(2+), reflecting closer interactions with TnC under these conditions. In reconstituted thin filaments, the environment of the AEDANS on Cys-96 was not greatly affected by Ca(2+), while the AEDANS on Cys-117 was more accessible but significantly less flexible as it moved away from actin and interacted strongly with TnC in the presence of Ca(2+). We used fluorescence resonance energy transfer (FRET) to measure distances between AEDANS on TnI Cys-96 or Cys-117 and 4-¿[(dimethylamino)phenyl]azo¿phenyl-4'-maleimide (DABmal) on actin Cys-374 in reconstituted thin filaments. In the absence of Ca(2+), the mean distances were 40.2 A for Cys-96 and 35.2 A for Cys-117. In the presence of Ca(2+), Cys-96 moved away from actin Cys-374 by approximately 3.6 A, while Cys-117 moved away by approximately 8 A. This suggests the existence of a flexible "hinge" region near the middle of TnI, allowing amino acid residues in the N-terminal half of TnI to interact with TnC in a Ca(2+)-independent manner, while the C-terminal half of TnI binds to actin in the absence of Ca(2+) or to TnC in the presence of Ca(2+). This is the first report to demonstrate structural movement of the inhibitory region of TnI in the thin filament.
在肌肉细肌丝中，肌钙蛋白I（TnI）的抑制区域（96 - 117位氨基酸残基）被认为在有Ca(2+)存在时与肌钙蛋白C（TnC）相互作用，而在无Ca(2+)时与肌动蛋白相互作用。为了更好地理解这些相互作用，我们制备了含有单个半胱氨酸-96或半胱氨酸-117的突变型TnI，并使用硫醇特异性荧光探针N-(碘乙酰基)-N'-(1-磺基-5-萘基)乙二胺（IAEDANS）对其进行标记。我们通过测量丙烯酰胺对荧光的猝灭程度和寿命分辨各向异性，来表征在有和无Ca(2+)存在时TnI上IAEDANS标记的微环境。在肌钙蛋白-原肌球蛋白（Tn-Tm）复合物中，在有Ca(2+)存在时，半胱氨酸-96和半胱氨酸-117上的IAEDANS标记与溶剂的接触较少且灵活性较低，这反映了在这些条件下与TnC的相互作用更紧密。在重构的细肌丝中，半胱氨酸-96上IAEDANS的环境受Ca(2+)的影响不大，而半胱氨酸-117上的IAEDANS更容易接近，但随着它远离肌动蛋白并在有Ca(2+)存在时与TnC强烈相互作用，其灵活性显著降低。我们使用荧光共振能量转移（FRET）来测量重构细肌丝中TnI半胱氨酸-96或半胱氨酸-117上的IAEDANS与肌动蛋白半胱氨酸-374上的4-[(二甲基氨基)苯基]偶氮苯基-4'-马来酰亚胺（DABmal）之间的距离。在无Ca(2+)时，半胱氨酸-96的平均距离为40.2 Å，半胱氨酸-117为35.2 Å。在有Ca(2+)时，半胱氨酸-96远离肌动蛋白半胱氨酸-374约3.6 Å，而半胱氨酸-117远离约8 Å。这表明在TnI中部附近存在一个灵活的“铰链”区域，使得TnI N端一半的氨基酸残基能够以不依赖Ca(2+)的方式与TnC相互作用，而TnI的C端一半在无Ca(2+)时与肌动蛋白结合，或在有Ca(2+)时与TnC结合。这是首次报道在细肌丝中TnI抑制区域的结构运动。
