Van Eyk J E, Caday-Malcolm R A, Yu L, Irvin R T, Hodges R S
Department of Biochemistry and Medical Research Council of Canada Group in Protein Structure and Function, University of Alberta, Edmonton, Canada.
Protein Sci. 1995 Apr;4(4):781-90. doi: 10.1002/pro.5560040418.
Multiple-component regulatory protein systems function through a generalized mechanism where a single regulatory protein or ligand binds to a variety of receptors to modulate specific functions in a physiologically sensitive context. Muscle contraction is regulated by the interaction of actin with troponin I (TnI) or myosin in a Ca(2+)-sensitive manner. Actin utilizes a single binding domain (residues 1-28) to bind to residues 104-115 of TnI (Van Eyk JE, Sönnichsen FD, Sykes BD, Hodges RS, 1991, In: Rüegg JC, ed, Peptides as probes in muscle research, Heidelberg, Germany: Springer-Verlag, pp 15-31) and to myosin subfragment 1 (S1, an enzymatic fragment of myosin containing both the actin and ATP binding sites) (Van Eyk JE, Hodges RS, 1991, Biochemistry 30:11676-11682) in a Ca(2+)-sensitive manner. We have utilized an anti-TnI peptide (104-115) monoclonal antibody, Mab B4, that binds specifically to TnI, to image the common binding domain of actin and thus mimic the activity of actin including activation of the S1 ATPase activity and TnI-mediated regulation of the S1 ATPase. Mab B4 has also been utilized to identify a receptor binding domain on myosin (residues 633-644) that is recognized by actin. Interestingly, Mab B4 binds to the native protein receptors TnI and S1 with relative affinities of 100- and 25,000-fold higher than the binding affinity to the 12-residue peptide immunogen. Thus, anti-peptide monoclonal antibodies prepared against a receptor binding domain can mimic the ligand binding domain and be utilized as a powerful tool for the detailed analysis of complex multiple-component regulatory systems.
多组分调节蛋白系统通过一种普遍机制发挥作用，即单个调节蛋白或配体与多种受体结合，在生理敏感环境中调节特定功能。肌肉收缩受肌动蛋白与肌钙蛋白I（TnI）或肌球蛋白以Ca(2+)敏感方式相互作用的调节。肌动蛋白利用单个结合结构域（第1至28位残基）以Ca(2+)敏感方式与TnI的第104至115位残基结合（Van Eyk JE、Sönnichsen FD、Sykes BD、Hodges RS，1991年，载于：Rüegg JC编，《肽作为肌肉研究中的探针》，德国海德堡：施普林格出版社，第15 - 31页）以及与肌球蛋白亚片段1（S1，肌球蛋白的一个酶片段，包含肌动蛋白和ATP结合位点）结合（Van Eyk JE、Hodges RS，1991年，《生物化学》30:11676 - 11682）。我们利用了一种抗TnI肽（104 - 115）单克隆抗体Mab B4，它特异性结合TnI，以成像肌动蛋白的共同结合结构域，从而模拟肌动蛋白的活性，包括激活S1 ATP酶活性以及TnI介导的对S1 ATP酶的调节。Mab B4还被用于鉴定肌球蛋白上一个被肌动蛋白识别的受体结合结构域（第633至644位残基）。有趣的是，Mab B4与天然蛋白受体TnI和S1的结合亲和力分别比与12残基肽免疫原的结合亲和力高100倍和25000倍。因此，针对受体结合结构域制备的抗肽单克隆抗体可以模拟配体结合结构域，并用作详细分析复杂多组分调节系统的有力工具。
