Das Nilimesh, Yadav Sandeep, Negi Kuldeep Singh, Tarif Ejaj, Sen Pratik
Department of Chemistry, Indian Institute of Technology Kanpur, Kanpur 208 016, UP, India.
BBA Adv. 2022 Jan 12;2:100041. doi: 10.1016/j.bbadva.2022.100041. eCollection 2022.
Proteins are dynamic entity with various molecular motions at different timescale and length scale. Molecular motions are crucial for the optimal function of an enzyme. It seems intuitive that these motions are crucial for optimal enzyme activity. However, it is not easy to directly correlate an enzyme's dynamics and activity due to biosystems' enormous complexity. amongst many factors, structure and dynamics are two prime aspects that combinedly control the activity. Therefore, having a direct correlation between protein dynamics and activity is not straightforward. Herein, we observed and correlated the structural, functional, and dynamical responses of an industrially crucial proteolytic enzyme, bromelain with three versatile classes of chemicals: GnHCl (protein denaturant), sucrose (protein stabilizer), and Ficoll-70 (macromolecular crowder). The only free cysteine (Cys-25 at the active-site) of bromelain has been tagged with a cysteine-specific dye to unveil the structural and dynamical changes through various spectroscopic studies both at bulk and at the single molecular level. Proteolytic activity is carried out using casein as the substrate. GnHCl and sucrose shows remarkable structure-dynamics-activity relationships. Interestingly, with Ficoll-70, structure and activity are not correlated. However, microsecond dynamics and activity are beautifully correlated in this case also. Overall, our result demonstrates that bromelain dynamics in the microsecond timescale around the active-site is probably a key factor in controlling its proteolytic activity.
蛋白质是动态实体，在不同的时间尺度和长度尺度上具有各种分子运动。分子运动对于酶的最佳功能至关重要。这些运动对于最佳酶活性至关重要，这似乎是直观的。然而，由于生物系统的巨大复杂性，直接关联酶的动力学和活性并不容易。在许多因素中，结构和动力学是共同控制活性的两个主要方面。因此，蛋白质动力学与活性之间的直接关联并不简单。在此，我们观察并关联了一种工业上至关重要的蛋白水解酶菠萝蛋白酶与三类通用化学品（盐酸胍（蛋白质变性剂）、蔗糖（蛋白质稳定剂）和聚蔗糖 -70（大分子拥挤剂））的结构、功能和动力学响应。菠萝蛋白酶唯一的游离半胱氨酸（活性位点的Cys-25）已用半胱氨酸特异性染料标记，以通过在整体和单分子水平上的各种光谱研究揭示结构和动力学变化。使用酪蛋白作为底物进行蛋白水解活性测定。盐酸胍和蔗糖显示出显著的结构 - 动力学 - 活性关系。有趣的是，对于聚蔗糖 -70，结构和活性不相关。然而，在这种情况下，微秒级动力学与活性也完美相关。总体而言，我们的结果表明，活性位点周围微秒时间尺度上的菠萝蛋白酶动力学可能是控制其蛋白水解活性的关键因素。
