Nanda Ananya, Nasker Sourya Subhra, Kushwaha Anoop K, Ojha Deepak Kumar, Dearden Albert K, Nayak Saroj K, Nayak Sasmita
School of Biotechnology, Kalinga Institute of Industrial Technology, Bhubaneswar, India.
School of Basic Sciences, Indian Institute of Technology Bhubaneswar, Bhubaneswar, India.
Front Bioeng Biotechnol. 2021 Dec 23;9:773303. doi: 10.3389/fbioe.2021.773303. eCollection 2021.
Protein splicing is a self-catalyzed event where the intervening sequence intein cleaves off, joining the flanking exteins together to generate a functional protein. Attempts have been made to regulate the splicing rate through variations in temperature, pH, and metals. Although metal-regulated protein splicing has been more captivating to researchers, metals were shown to only inhibit splicing reactions that confine their application. This is the first study to show the effect of nanoparticles (NPs) on protein splicing. We found that gold nanoparticles (AuNPs) of various sizes can increase the splicing efficiency by more than 50% and the N-terminal cleavage efficiency by more than 45% in SufB precursor protein. This study provides an effective strategy for engineering splicing-enhanced intein platforms. UV-vis absorption spectroscopy, isothermal titration calorimetry (ITC), and transmission electron microscopy (TEM) confirmed AuNP interaction with the native protein. Quantum mechanics/molecular mechanics (QM/MM) analysis suggested a significant reduction in the energy barrier at the N-terminal cleavage site in the presence of gold atom, strengthening our experimental evidence on heightened the N-terminal cleavage reaction. The encouraging observation of enhanced N-terminal cleavage and splicing reaction can have potential implementations from developing a rapid drug delivery system to designing a contemporary protein purification system.
蛋白质剪接是一个自我催化的过程，其中居间序列内含肽会裂解下来，将侧翼外显肽连接在一起，从而产生一个功能性蛋白质。人们已尝试通过改变温度、pH值和金属来调节剪接速率。尽管金属调控的蛋白质剪接更吸引研究人员，但金属仅被证明会抑制剪接反应，限制了它们的应用。这是第一项展示纳米颗粒（NPs）对蛋白质剪接影响的研究。我们发现，各种尺寸的金纳米颗粒（AuNPs）可使SufB前体蛋白的剪接效率提高50%以上，N端裂解效率提高45%以上。本研究为构建剪接增强的内含肽平台提供了一种有效策略。紫外可见吸收光谱、等温滴定量热法（ITC）和透射电子显微镜（TEM）证实了AuNP与天然蛋白质的相互作用。量子力学/分子力学（QM/MM）分析表明，在有金原子存在的情况下，N端裂解位点的能垒显著降低，这加强了我们关于增强N端裂解反应的实验证据。N端裂解和剪接反应增强这一令人鼓舞的发现，在从开发快速药物递送系统到设计现代蛋白质纯化系统等方面可能具有潜在应用。
