Probing calmodulin-NO synthase interactions via site-specific infrared spectroscopy: an introductory investigation
Probing calmodulin-NO synthase interactions via site-specific infrared spectroscopy: an introductory investigation
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DOI：
10.1007/s00775-024-02046-0
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发表时间：
2024-04-05
影响因子：
3
通讯作者：
Feng，Changjian
中科院分区：
文献类型：
--
作者：
Singh，Swapnil;Gyawali，Yadav Prasad;Feng，Changjian
钙调素（CaM）与一氧化氮合酶（NOS）的加氧酶和还原酶结构域之间的连接体结合，以调节功能构象动力学。结构域间界面上的特定残基引导结构域-结构域对接以促进NOS中的电子转移。值得注意的是，钙调素和含血红素的NOS加氧酶结构域之间的对接界面是异构体特异性的，这才刚刚开始研究。为了进一步了解不同的CaM-NOS对接相互作用的红外光谱，我们引入了一个氰基作为频率分辨振动探针到钙调素单独和当与全长和双结构域加氧酶/FMN结构的诱导型一氧化氮合酶亚型（iNOS）。由于单独重组iNOS蛋白的不稳定性，通过琥珀抑制与iNOS结合的CaM，用对氰基-L-苯丙氨酸（CNF）进行位点特异性的选择性标记已经通过蛋白共表达来实现。我们在第108位残基上引入CNF，这是在推定的CaM-heme（NOS）对接界面处，CNF也被引入到第29位残基上，这是远离对接界面的。FT IR数据显示108位点对CaM-NOS复合物的形成敏感，而29位点对其与iNOS蛋白或肽的结合不敏感。此外，在残基108处的IR谱带变窄表明C-3 N探针经历了更有限的环境分布，表明侧链限制对于与iNOS的复合物是明显的。这项初步工作为NOS蛋白质对接状态的结构动力学的残基特异性表征奠定了基础。图形摘要
Calmodulin (CaM) binds to a linker between the oxygenase and reductase domains of nitric oxide synthase (NOS) to regulate the functional conformational dynamics. Specific residues on the interdomain interface guide the domain-domain docking to facilitate the electron transfer in NOS. Notably, the docking interface between CaM and the heme-containing oxygenase domain of NOS is isoform specific, which is only beginning to be investigated. Toward advancing understanding of the distinct CaM–NOS docking interactions by infrared spectroscopy, we introduced a cyano-group as frequency-resolved vibrational probe into CaM individually and when associated with full-length and a bi-domain oxygenase/FMN construct of the inducible NOS isoform (iNOS). Site-specific, selective labeling withp-cyano-l-phenylalanine (CNF) by amber suppression of CaM bound to the iNOS has been accomplished by protein coexpression due to the instability of recombinant iNOS protein alone. We introducedCNF at residue 108, which is at the putative CaM–heme (NOS) docking interface.CNF was also introduced at residue 29, which is distant from the docking interface. FT IR data show that the 108 site is sensitive to CaM–NOS complex formation, while insensitivity to its association with the iNOS protein or peptide was observed for the 29 site. Moreover, narrowing of the IR bands at residue 108 suggests the C≡N probe experiences a more limited distribution of environments, indicating side chain restriction apparent for the complex with iNOS. This initial work sets the stage for residue-specific characterizations of structural dynamics of the docked states of NOS proteins.Graphical abstract