肌钙蛋白C(Tropnin C)的提纯与鉴定

<正> 肌钙蛋白C(TnC)是肌钙蛋白复合体的钙结合亚基,系骨骼肌和心肌收缩系统的触发因子。研究表明TnC与钙调素(CaM)类似,具有EF手结构和四个特殊的钙结合区。TnC与Ca~(2+)、Mg~(2+)等金属离子的结合可发生构型变化及由此导致的体内一系列酶活性等生理功能的变化。提纯TnC是进行其诸方面研究的基础。     

快速骨骼肌型肌钙蛋白Ⅰ的研究进展

快速骨骼肌型肌钙蛋白Ⅰ(fast skeletal muscle troponin Ⅰ,fsTnI),即肌钙蛋白Ⅰ2(TNNI2),是肌钙蛋白(troponin,Tn)三元复合物中的抑制亚基存在于快速骨骼肌中的一砷组织亚型,是调节脊推动物横纹肌收缩的重要蛋白质。该着重回顾fsTnⅠ在蛋白质和基因结构等方面的研究,对近年来有关fsTnⅠ抑制肿瘤血管生成的研究,以及对其可能以核受体辅活化子身份参与基因表达调控的潜质做简要介绍。     

心肌肌钙蛋白Ⅰ的磷酸化与非磷酸化调节

由于心肌肌钙蛋白复合体Ⅰ亚基(Troponin Ⅰ,TnⅠ)特殊的分子结构,使其在心肌收缩过程中起"分子开关"的重要作用.心肌TnⅠ具有6个磷酸化位点,第23/24位丝氨酸残基可被蛋白激酶A(PKA)、蛋白激酶D(PKD)和蛋白激酶G(PKG)磷酸化,发挥正性肌力作用;第43/45位丝氨酸残基以及第144位酪氨酸残基可被蛋白激酶C(PKC)磷酸化,可能主要起负性肌力作用;蛋白激活激酶(PAK)磷酸化第149位丝氨酸残基后的作用尚待探明.另外,经蛋白水解酶calpain降解含磷酸化位点的片段,产生去磷酸化作用;亦可通过降解一些特定片段来改变TnⅠ空间构象,引起非磷酸化调节作用.     

线粒体呼吸链复合体Ⅰ

线粒体呼吸链复合体Ⅰ(简称复合体Ⅰ)是呼吸链电子传递的起始复合体,作为电子传递过程的限速酶,复合体Ⅰ的分子量远大于其余的四个呼吸链复合体。复合体Ⅰ相关的疾病发生除了与40余个复合体Ⅰ组成亚基的突变相关外,还同参与其组装的多个组装因子存在密切联系。该文对复合体I的结构以及参与调控复合体Ⅰ组装的各类组装因子进行了综述,旨在为全面了解复合体Ⅰ相关疾病的发生提供具体参考。     

线粒体呼吸链复合体Ⅰ

线粒体呼吸链复合体Ⅰ（简称复合体Ⅰ）是呼吸链电子传递的起始复合体,作为电子传递过程的限速酶,复合体Ⅰ的分子量远大于其余的四个呼吸链复合体。复合体Ⅰ相关的疾病发生除了与40余个复合体Ⅰ组成亚基的突变相关外,还同参与其组装的多个组装因子存在密切联系。该文对复合体I的结构以及参与调控复合体Ⅰ组装的各类组装因子进行了综述,旨在为全面了解复合体Ⅰ相关疾病的发生提供具体参考。     

跨膜Ca~(2+)梯度对肌质网Ca~3+-ATP酶调节的特异性

我们曾报道跨膜Ca~(2+)梯度可通过膜脂影响肌质网Ca~(2+)-ATP 酶的构象和活性。本文就跨膜Ca~(2+)梯度对肌质网Ca~(2+)-ATP 酶的调节是否具有特异性作进一步研究。结果表明这种特异性表现在两方面:一是跨膜Ca~(2+)梯度对肌质网Ca~(2+)-ATP 酶功能的调节不能归结于跨膜Ca~(2+)浓度梯度所导致的膜电位的作用,离子载体FCCP 可消除跨膜电位但并不影响肌质网Ca~(2+)-ATP 酶的活力;二是其它二价金属离子如Sr~(2+)的跨膜梯度对肌质网Ca~(2+)-ATP 酶活力基本无影响。荧光偏振系列探剂n-AS 测定的结果表明跨膜Ca~(2+)与Sr~(2+)梯度对嵌有Ca~(2+)-ATP 酶的脂酶体的中部流动性的影响有较大差异。而Ca~(2+)-ATP 酶的Ca~(2+)结合位点正处于脂双层中部,这进一步提示膜脂参与了跨膜Ca~(2+)梯度对Ca~(2+)-ATP 酶的调节作用。     

核酸适配体生物传感器对心肌肌钙蛋白Ⅰ的检测研究

核酸适配体生物传感器是利用固定在电极表面的适配子与被测溶液中心肌肌钙蛋白Ⅰ(cTnⅠ)发生特异性结合,从而达到检测的目的.我们对玻碳电极进行阳极氧化、氨基化修饰,通过碳二亚胺盐酸盐(carbodiimide hydrochloride,EDC)、N-羟基琥珀酰亚胺(N-hydroxysuccinimide,NHS)活化作用将适配子结合在电极表面.cTnⅠ最佳检测范围是0.05~5 nmol/L,最低检测限为0.05 nmol/L,检测时间为5 min.     

非损伤微测技术实时检测海马脑片跨膜钙离子流

脑内β-淀粉样蛋白(amyloid-βprotein,Aβ)的聚集是阿尔茨海默病(Alzheimer’s disease,AD)的重要病理特征。Aβ的神经毒性作用机制与其扰乱神经元Ca~(2+)稳态有密切关系。非损伤微测技术(non-invasive micro-test technique,NMT)是近年发展起来的一种利用Fick第一扩散定律和Nernst方程,通过非接触方式检测膜外扩散电位获取离子跨膜流速的最新技术手段。本研究在C57BL/6小鼠海马脑片,利用NMT首次检测了Aβ对谷氨酸(Glu)诱发的Ca~(2+)内流以及细胞外低钙引起的Ca~(2+)外排的影响,并初步探讨了Aβ扰乱神经元Ca~(2+)稳态的相关机制。结果显示:(1)急性给予Glu可诱发海马脑片CA1区神经元产生起始快、继而缓慢衰减的持续性内向Ca~(2+)流;(2)Aβ预处理浓度依赖性地增强海马神经元对Glu的反应性,显著提高给药后5 min内Ca~(2+)内流的平均流速,而NMDA受体拮抗剂D-APV可有效阻断Aβ对神经元Glu反应的这种易化作用;(3)用低钙人工脑脊液急性灌流脑片可引起海马CA1区神经元产生持续的外向跨膜Ca~(2+)流,其大部分可被特异性Na+/Ca~(2+)交换体抑制剂KB-R7943所阻断;(4)Aβ预处理可部分抑制低钙人工脑脊液引起的Ca~(2+)外排。这些结果表明:Aβ引起的细胞内Ca~(2+)超载不仅涉及到Ca~(2+)内流增加,也与其对Ca~(2+)外排的抑制有关;Aβ易化Glu的兴奋毒作用主要是通过NMDA受体介导的,其抑制Ca~(2+)外排的靶点主要是Na+/Ca~(2+)交换体。NMT具有操作相对简单、实时获取结果、非损伤的优点,适用于脑片Ca~(2+)内流和Ca~(2+)外排的长时间测定。因此,本研究不仅为解释Aβ所致Ca~(2+)超载的神经毒性机制提供了新的实验证据,也为开展跨膜Ca~(2+)信号转导机制的脑研究提供了新的技术方法。     

限制性内切酶NotⅠ提纯的新工艺

目前有关限制性内切酶NotⅠ的性质特征及功能机制等方面的研究日渐增多,但商品化NotⅠ及某些限制性内切酶的价格依然居高不下,其主要原因在于表达量低、提纯程序繁琐、得率低等问题的存在。为探索限制性内切酶NotⅠ提纯的新工艺,从豚鼠耳炎诺卡菌(Nocardia otitidis-caviarum)中克隆出限制性内切酶NotⅠ的基因并使之在大肠杆菌中高效表达。首先将由成团肠杆菌(Enterobacter agglomerans)中克隆所得甲基化酶EagⅠM(EagⅠ methylase gene)基因连接到pBR322载体上,转化大肠杆菌ER2566,将豚鼠耳炎诺卡菌中克隆所得的限制性内切酶NotⅠR(NotⅠrestriction endonuclease gene)基因连接到表达载体pACYC184-PT₇上,将此重组质粒转化到上述已转入甲基化重组质粒pBR322-EagⅠM的ER2566中,构建成NotⅠ蛋白表达菌ER2566 。重组工程菌经IPTG诱导可表达限制性内切酶NotⅠ,并对诱导条件进行优化使之以可溶形式高效表达。应用KTA purifier 100蛋白纯化系统,对纯化工艺进行创新,通过DEAE Sephrose FF离子交换层析、phenyl HP疏水层析和Superdex 75 10/300 GL分子筛层析对蛋白进行提纯。纯化后NotⅠ经酶活力及纯度鉴定,其比活力为1.37×10⁶U/mg,提纯35倍,得率为17.8％,产量达9.8×10⁶ Units /g wet cell,提纯时间缩减为原来的1/10,在产量和效率上较以前报道均有很大提高。该纯化工艺的新方法,为实验室制备及工业化生产Ⅱ型限制性内切酶提供了进一步的借鉴。且该酶的成功获得为后续研究提供了材料,为更多新发现内切酶的成功克隆提供了参考。     

心肌肌钙蛋白Ⅰ的分子印迹电化学生物传感器

应用分子印迹技术,以邻苯二胺和对苯二酚为功能单体,心肌肌钙蛋白Ⅰ(cTnI)为模板分子,在pH 7.0磷酸盐缓冲液中,利用循环伏安法在玻碳电极表面聚合形成了分子印迹膜.该分子膜对cTnI有特异性识别作用,在0.01～2.00 μg/mL的范围内,cTnI的浓度与氧化峰电流的变化呈线性关系,检测下限为2 ng/mL,响应时间为15 min.该分子印迹传感器具有制备简单、特异性及稳定性好等优点.     

Calcium- and magnesium-dependent interactions between the C-terminus of troponin I and the N-terminal, regulatory domain of troponin C

The muscle thin filament protein troponin (Tn) regulates contraction of vertebrate striated muscle by conferring Ca2+ sensitivity to the interaction of actin and myosin. Troponin C (TnC), the Ca2+ binding subunit of Tn contains two homologous domains and four divalent cation binding sites. Two structural sites in the C-terminal domain of TnC bind either Ca2+ or Mg2+, and two regulatory sites in the N-terminal domain are specific for Ca2+. Interactions between TnC and the inhibitory Tn subunit troponin I (TnI) are of central importance to the Ca2+ regulation of muscle contraction and have been intensively studied. Much remains to be learned, however, due mainly to the lack of a three-dimensional structure for TnI. In particular, the role of amino acid residues near the C-terminus of TnI is not well understood. In this report, we prepared a mutant TnC which contains a single Trp-26 residue in the N-terminal, regulatory domain. We used fluorescence lifetime and quenching measurements to monitor Ca2+- and Mg2+-dependent changes in the environment of Trp-26 in isolated TnC, as well as in binary complexes of TnC with a Trp-free mutant of TnI or a truncated form of this mutant, TnI(1-159), which lacked the C-terminal 22 amino acid residues of TnI. We found that full-length TnI and TnI(1-159) affected Trp-26 similarly when all four binding sites of TnC were occupied by Ca2+. When the regulatory Ca2+-binding sites in the N-terminal domain of TnC were vacant and the structural sites in the C-terminal domain of were occupied by Mg2+, we found significant differences between full-length TnI and TnI(1-159) in their effect on Trp-26. Our results provide the first indica- tion that the C-terminus of TnI may play an important role in the regulation of vertebrate striated muscle through Ca2+-dependent interactions with the regula- tory domain of TnC.     

The calcium-induced switch in the troponin complex probed by fluorescent mutants of troponin I.

The Ca2+-induced transition in the troponin complex (Tn) regulates vertebrate striated muscle contraction. Tn was reconstituted with recombinant forms of troponin I (TnI) containing a single intrinsic 5-hydroxytryptophan (5HW). Fluorescence analysis of these mutants of TnI demonstrate that the regions in TnI that respond to Ca2+ binding to the regulatory N-domain of TnC are the inhibitory region (residues 96-116) and a neighboring region that includes position 121. Our data confirms the role of TnI as a modulator of the Ca2+ affinity of TnC; we show that point mutations and incorporation of 5HW in TnI can affect both the affinity and the cooperativity of Ca2+ binding to TnC. We also discuss the possibility that the regulatory sites in the N-terminal domain of TnC might be the high affinity Ca2+-binding sites in the troponin complex.     

Calcium-dependent movement of troponin I between troponin C and actin as revealed by spin-labeling EPR

We measured EPR spectra from a spin label on the Cys133 residue of troponin I (TnI) to identify Ca(2+)-induced structural states, based on sensitivity of spin-label mobility to flexibility and tertiary contact of a polypeptide. Spectrum from Tn complexes in the -Ca(2+) state showed that Cys133 was located at a flexible polypeptide segment (rotational correlation time tau=1.9ns) that was free from TnC. Spectra of both Tn complexes alone and those reconstituted into the thin filaments in the +Ca(2+) state showed that Cys133 existed on a stable segment (tau=4.8ns) held by TnC. Spectra of reconstituted thin filaments (-Ca(2+) state) revealed that slow mobility (tau=45ns) was due to tertiary contact of Cys133 with actin, because the same slow mobility was found for TnI-actin and TnI-tropomyosin-actin filaments lacking TnC, T or tropomyosin. We propose that the Cys133 region dissociates from TnC and attaches to the actin surface on the thin filaments, causing muscle relaxation at low Ca(2+) concentrations.     

Conformational changes induced in troponin I by interaction with troponin T and actin/tropomyosin.

Troponin I (TnI) is the inhibitory component of the striated muscle Ca2+ regulatory protein troponin (Tn). The other two components of Tn are troponin C (TnC), the Ca2+-binding component, and troponin T (TnT), the tropomyosin-binding component. We have used limited chymotryptic digestion to probe the local conformation of TnI in the free state, the binary TnC*TnI complex, the ternary TnC*. TnI*TnT (Tn) complex, and in the reconstituted Tn*tropomyosin*F-actin filament. The digestion of TnI alone or in the TnC*TnI complex produced initially two major fragments via a cleavage of the peptide bond between Phe100 and Asp101 in the so-called inhibitory region. In the ternary Tn complex cleavage occurred at a new site between Leu140 and Lys141. In the absence of Ca2+ this was followed by digestion of the 1-140 fragment at Leu122 and Met116. In the reconstituted thin filament the same fragments as in the case of the ternary complex were produced, but the rate of digestion was slower in the absence than in the presence of Ca2+. These results indicate firstly that in both free TnI and TnI complexed with TnC there is an exposed and flexible site in the inhibitory region. Secondly, TnT affects the conformation of TnI in the inhibitory region and also in the region that contains the 140-141 bond. Thirdly, the 140-141 region of TnI is likely to interact with actin in the reconstituted thin filament when Ca2+ is absent. These findings are discussed in terms of the role of TnI in the mechanism of thin filament regulation, and in light of our previous results [Y. Luo, J.-L. Wu, J. Gergely, T. Tao, Biochemistry 36 (1997) 13449-13454] on the global conformation of TnI.     

Functional importance of Ca2+-deficient N-terminal lobe of molluscan troponin C in troponin regulation

Ca(2+)-binding sites I and II in the N-terminal lobe of molluscan troponin C (TnC) have lost the ability to bind Ca(2+) due to substitutions of the amino acid residues responsible for Ca(2+) liganding. To evaluate the functional importance of the Ca(2+)-deficient N-terminal lobe in the Ca(2+)-regulatory function of molluscan troponin, we constructed chimeric TnCs comprising the N-terminal lobes from rabbit fast muscle and squid mantle muscle TnCs and the C-terminal lobe from akazara scallop TnC, TnC(RA), and TnC(SA), respectively. We characterized their biochemical properties as compared with those of akazara scallop wild-type TnC (TnC(AA)). According to equilibrium dialysis using (45)Ca(2+), TnC(RA), and TnC(SA) bound stoichiometrically 3 mol Ca(2+)/mol and 1 mol Ca(2+)/mol, respectively, as expected from their primary structures. All the chimeric TnCs exhibited difference-UV-absorption spectra at around 280-290 nm upon Ca(2+) binding and formed stable complexes with akazara scallop troponin I, even in the presence of 6M urea, if Ca(2+) was present. However, when the troponin complexes were constructed from chimeric TnCs and akazara scallop troponin T and troponin I, they showed different Ca(2+)-regulation abilities from each other depending on the TnC species. Thus, the troponin containing TnC(SA) conferred as high a Ca(2+) sensitivity to Mg-ATPase activity of rabbit actomyosin-akazara scallop tropomyosin as did the troponin containing TnC(AA), whereas the troponin containing TnC(RA) conferred virtually no Ca(2+) sensitivity. Our findings indicate that the N-terminal lobe of molluscan TnC plays important roles in molluscan troponin regulation, despite its inability to bind Ca(2+).     

Static and kinetic studies on rabbit skeletal muscle troponin

Fluorescence titration curves of 2-[4'-iodoacetamido)anilino)naphthalene-6-sulfonic acid-labeled troponin (IAANS-labeled Tn) and troponin-1-anilinonaphthalene-8-sulfonic acid (Tn-ANS) complex indicated that the fluorescent moiety, IAANS or ANS, detects conformational change of troponin I (TnI) or Tn due to the Ca2+ binding or removal reaction with the low affinity Ca2+-binding sites of troponin C (TnC) component. A fluorescence stopped-flow study showed that the kinetic behavior of IAANS-labeled Tn reflects a change in state of the TnI component induced by the Ca2+ binding or removal reaction with the low affinity Ca2+-binding sites of TnC component. The state change of TnI induced by the Ca2+ binding was complete within the instrumental dead time. On the other hand, that induced by the Ca2+ removal had a rate constant of around 13 s-1. ANS, which is noncovalently bound to Tn, reflects the kinetic properties of both the TnI component and the low affinity Ca2+-binding region of TnC component. The fluorescence intensity change of ANS induced by Ca2+ binding to the low affinity Ca2+-binding sites of TnC was complete within the instrumental dead time, while that induced by the Ca2+ removal from the same sites was biphasic. The rate constants of the biphasic process were found to be 62 +/- 7 s-1 and 16 +/- 4 s-1. The former value corresponds to the rate constant of the Ca2+ removal reaction from the low affinity Ca2+-binding sites of TnC component, and the latter value to the rate constant observed in the case of IAANS-labeled Tn. Based on these experimental results and on the discussion in our previous paper (Iio, T. & Kondo, H. (1981) J. Biochem. 90, 163-175), we have refined the two-way information-transfer mechanism which we previously proposed in order to explain the biological function of Tn.     

The role of the NH(2)- and COOH-terminal domains of the inhibitory region of troponin I in the regulation of skeletal muscle contraction.

The role of the inhibitory region of troponin (Tn) I in the regulation of skeletal muscle contraction was studied with three deletion mutants of its inhibitory region: 1) complete (TnI-(Delta96-116)), 2) the COOH-terminal domain (TnI-(Delta105-115)), and 3) the NH(2)-terminal domain (TnI-(Delta95-106)). Measurements of Ca(2+)-regulated force and relaxation were performed in skinned skeletal muscle fibers whose endogenous TnI (along with TnT and TnC) was displaced with high concentrations of added troponin T. Reconstitution of the Tn-displaced fibers with a TnI.TnC complex restored the Ca(2+) sensitivity of force; however, the levels of relaxation and force development varied. Relaxation of the fibers (pCa 8) was drastically impaired with two of the inhibitory region deletion mutants, TnI-(Delta96-116).TnC and TnI-(Delta105-115).TnC. The TnI-(Delta95-106).TnC mutant retained approximately 55% relaxation when reconstituted in the Tn-displaced fibers. Activation in skinned skeletal muscle fibers was enhanced with all TnI mutants compared with wild-type TnI. Interestingly, all three mutants of TnI increased the Ca(2+) sensitivity of contraction. None of the TnI deletion mutants, when reconstituted into Tn, could inhibit actin-tropomyosin-activated myosin ATPase in the absence of Ca(2+), and two of them (TnI-(Delta96-116) and TnI-(Delta105-115)) gave significant activation in the absence of Ca(2+). These results suggest that the COOH terminus of the inhibitory region of TnI (residues 105-115) is much more critical for the biological activity of TnI than the NH(2)-terminal region, consisting of residues 95-106. Presumably, the COOH-terminal domain of the inhibitory region of TnI is a part of the Ca(2+)-sensitive molecular switch during muscle contraction.     

Photocrosslinking of benzophenone-labeled single cysteine troponin I mutants to other thin filament proteins

The interaction sites of rabbit skeletal troponin I (TnI) with troponin C (TnC), troponin T (TnT), tropomyosin (Tm) and actin were mapped systematically using nine single cysteine residue TnI mutants with mutation sites at positions 6, 48, 64, 89, 104, 121, 133, 155 or 179 (TnI6, TnI48 etc.). Each mutant was labeled with the heterobifunctional photocrosslinker 4-maleimidobenzophenone (BP-Mal), and incorporated into the TnI.TnC binary complex, the TnI.TnC.TnT ternary troponin (Tn) complex, and the Tn.Tm.F-actin synthetic thin filament. Photocrosslinking reactions carried out in the presence and absence of Ca(2+) yielded the following results: (1) BP-TnI6 photocrosslinked primarily to TnC with a small degree of Ca(2+)-dependence in all the complex forms. (2) BP-TnI48, TnI64 and TnI89 photocrosslinked to TnT with no Ca(2+)-dependence. Photocrosslinking to TnC was reduced in the ternary versus the binary complex. BP-TnI89 also photocrosslinked to actin with higher yields in the absence of Ca(2+) than in its presence. (3) BP-TnI104 and TnI133 photocrosslinked to actin with much higher yields in the absence than in the presence of Ca(2+). (4) BP-TnI121 photocrosslinked to TnC with a small degree of Ca(2+)-dependence, and did not photocrosslink to actin. (5) BP-TnI155 and TnI179 photocrosslinked to TnC, TnT and actin, but all with low yields. All the labeled mutants photocrosslinked to TnC with varying degrees of Ca(2+)-dependence, and none to Tm. These results, along with those published allowed us to construct a structural and functional model of TnI in the Tn complex: in the presence of Ca(2+), residues 1-33 of TnI interact with the C-terminal domain hydrophobic cleft of TnC, approximately 48-89 with TnT, approximately 90-113 with TnC's central helix, approximately 114-125 with TnC's N-terminal domain hydrophobic cleft, and approximately 130-150 with TnC's A-helix. In the absence of Ca(2+), residues approximately 114-125 move out of TnC's N-terminal domain hydrophobic cleft and trigger the movements of residues approximately 89-113 and approximately 130-150 away from TnC and towards actin.     

Troponin I enhances acidic pH-induced depression of Ca2+ binding to the regulatory sites in skeletal troponin C

Inhibition of muscle force development by acidic pH is a well known phenomenon, yet the exact mechanism by which a decrease in pH inhibits the Ca2+-activated force in striated myofilaments remains poorly understood. Whether or not the deactivation by acidic pH involves direct competition between Ca2+ and protons for regulatory binding sites on fast skeletal troponin C (TnC) or whether other proteins in thin filament regulation are important remains unclear. We measured the effects of acidic pH on Ca2+-dependent fluorescent changes in TnC labeled with the probe danzylaziridine (Danz), which reports Ca2+ binding to the regulatory (Ca2+-specific) sites. Measurements were also made with TnCDanz complexed with the inhibitory Tn unit, TnI, and in the whole Tn complex. Our results show that a drop in pH from 7.0 to 6.5 is associated with a 1.6-fold increase in the midpoint for the relation between free Ca2+ and Ca2+ binding to the regulatory sites on TnCDanz. However, when TnCDanz was present in its complex with either TnI alone or with TnI-TnT, the increase in midpoint free Ca2+ was increased by 3.5-fold. We tested whether this potentiation in the effect of acidic pH on Ca2+ binding to TnC is due to a pH-induced alteration in the binding of TnI to TnC. A decrease in pH from 7.0 to 6.5 was associated with a halving of the affinity of TnI for TnC. We also probed the effect of acidic pH on TnI. This was done (i) by measuring the intrinsic fluorescence of tryptophan residues in TnI alone and (ii) by measuring fluorescence of TnI (in the Tn complex) labeled at Cys-133 with 5-iodoacetamidofluorescein. A drop in pH from 7.0 to 6.5 was associated with a 15% decrease in intrinsic fluorescence and with a 30% decrease in the fluorescence of the 5-iodoacetamidofluorescein probe. We conclude, therefore, that while protons and Ca2+ may directly affect Ca2+ binding to regulatory sites on fast skeletal TnC, the effect of acidic pH on TnC Ca2+ binding is amplified in the TnI-TnC and Tn complexes by a pH-related effect on the affinity of TnI for TnC.     

A cross-linking study of the N-terminal extension of human cardiac troponin I

Phosphorylation of the unique N-terminal extension of cardiac troponin I (TnI) by PKA modulates Ca(2+) release from the troponin complex. The mechanism by which phosphorylation affects Ca(2+) binding, however, remains unresolved. To investigate this question, we have studied the interaction of a fragment of TnI consisting of residues 1-64 (I1-64) with troponin C (TnC) by isothermal titration microcalorimetry and cross-linking. I1-64 binds extremely tightly to the C-terminal domain of TnC and weakly to the N-terminal domain. Binding to the N-domain is weakened further by phosphorylation. Using the heterobifunctional cross-linker benzophenone-4-maleimide and four separate cysteine mutants of I1-64 (S5C, E10C, I18C, R26C), we have probed the protein-protein interactions of the N-terminal extension. All four I1-64 mutants cross-link to the N-terminal domain of TnC. The cross-linking is enhanced by Ca(2+) and reduced by phosphorylation. By introducing the same monocysteine mutations into full-length TnI, we were able to probe the environment of the N-terminal extension in intact troponin. We find that the full length of the extension lies in close proximity to both TnC and troponin T (TnT). Ca(2+) enhances the cross-linking to TnC. Cross-linking to both TnC and TnT is reduced by prior phosphorylation of the TnI. In binary complexes the mutant TnIs cross-link to both the isolated TnC N-domain and whole TnC. Cyanogen bromide digestion of the covalent TnI-TnC complex formed from intact troponin demonstrates that cross-linking is predominantly to the N-terminal domain of TnC.