College of Pharmacy, University of New Mexico, Albuquerque, NM 87131, USA.
Department of Chemistry and Chemical Biology, University of New Mexico, Albuquerque, NM 87131, USA.
J Inorg Biochem. 2024 Feb;251:112454. doi: 10.1016/j.jinorgbio.2023.112454. Epub 2023 Dec 11.
Neuronal nitric oxide synthase (nNOS) is regulated by phosphorylation in vivo, yet the underlying biochemical mechanisms remain unclear, primarily due to difficulty in obtaining milligram quantities of phosphorylated nNOS protein; detailed spectroscopic and rapid kinetics investigations require purified protein samples at a concentration in the range of hundreds microM. Moreover, the functional diversity of the nNOS isoform is linked to its splice variants. Also of note is that determination of protein phosphorylation stoichiometry remains as a challenge. To address these issues, this study first expanded a recent genetic code expansion approach to produce phosphorylated rat nNOSμ and nNOSα holoproteins through site-specific incorporation of phosphoserine (pSer) at residues 1446 and 1412, respectively; this site is at the C-terminal tail region, a NOS-unique regulatory element. A quantitative mass spectrometric approach was then developed in-house to analyze unphosphorylated peptides in phosphatase-treated and -untreated phospho-nNOS proteins. The observed pSer-incorporation efficiency consistently exceeded 80%, showing high pSer-incorporation efficiency. Notably, EPR spin trapping results demonstrate that under l-arginine-depleted conditions, pSer1412 nNOSα presented a significant reduction in superoxide generation, whereas pSer1446 nNOSμ exhibited the opposite effect, compared to their unphosphorylated counterparts. This suggests that phosphorylation at the C-terminal tail has a regulatory effect on nNOS uncoupling that may differ between variant forms. Furthermore, the methodologies for incorporating pSer into large, complex protein and quantifying the percentage of phosphorylation in recombinant purified protein should be applicable to other protein systems.
神经元型一氧化氮合酶 (nNOS) 在体内受到磷酸化调控，但潜在的生化机制仍不清楚，主要是因为难以获得毫克级别的磷酸化 nNOS 蛋白；详细的光谱和快速动力学研究需要在数百微摩尔范围内的浓度下使用纯化的蛋白样品。此外，nNOS 同工型的功能多样性与其剪接变体有关。还值得注意的是，蛋白质磷酸化化学计量的测定仍然是一个挑战。为了解决这些问题，本研究首先扩展了最近的遗传密码扩展方法，通过在残基 1446 和 1412 处分别特异性掺入磷酸丝氨酸 (pSer)，产生磷酸化的大鼠 nNOSμ 和 nNOSα 全蛋白；该位点位于 C 末端尾部区域，是 NOS 独特的调节元件。然后，我们开发了一种内部的定量质谱方法，用于分析磷酸酶处理和未处理的磷酸化 nNOS 蛋白中的未磷酸化肽。观察到的 pSer 掺入效率始终超过 80%，表明具有高的 pSer 掺入效率。值得注意的是，EPR 自旋捕获结果表明，在 l-精氨酸耗尽的条件下，pSer1412 nNOSα 生成超氧化物的能力显著降低，而 pSer1446 nNOSμ 则表现出相反的效果，与未磷酸化的对应物相比。这表明 C 末端尾部的磷酸化对 nNOS 解偶联具有调节作用，这种调节作用可能因变体形式而异。此外，将 pSer 掺入大型复杂蛋白并定量重组纯化蛋白中磷酸化百分比的方法应该适用于其他蛋白系统。
