Department of Chemistry, University of Delhi , Delhi 110 007, India.
Department of Chemistry, College of Science, Sultan Qaboos University , PO Box 36, PC 123 Muscat, Oman.
J Phys Chem B. 2017 Jul 13;121(27):6456-6470. doi: 10.1021/acs.jpcb.7b01776. Epub 2017 Jun 30.
Characterization of a protein in the context of its environment is of crucial importance for a complete understanding of its function. Although biophysical techniques provide powerful tools for studying the stability and activity of the enzyme in the presence of various cosolvents, an approach of combining both experimental techniques and molecular dynamic (MD) simulations may lead to the mechanistic insight into the interactions governing the stability of an enzyme. The knowledge of these interactions can be further utilized for range of modifications in the wild form of an enzyme for various pharmaceutical applications. Herein, we employed florescence, UV-visible, circular dichroism (CD), dynamic light scattering (DLS) study, and MD simulations for comprehensive understanding of stem bromelain (BM) in the presence of betaine, sarcosine, arginine, and proline. The thermal stability of BM in the presence of 1 M of osmolytes is found to be in order: proline > betaine > buffer > arginine > sarcosine. BM gets more preferentially hydrated in the presence of betaine and proline than in sarcosine and arginine. Nonetheless, MD simulations suggest that betaine, sarcosine, and arginine at 1 M interact with the active site of BM through H-bonding except proline which are responsible for more disruption of active site. The distances between the catalytic site residues are 1.6, 1.9, 4.3, 5.0, and 6.2 Å for BM in proline, buffer, betaine, arginine, and sarcosine at 1 M, respectively. To the best of our knowledge, this is the first report on detailed unequivocal evidence of denaturation and deactivation of BM in the presence of methylamines and amino acids.
在环境背景下对蛋白质进行特征描述对于全面了解其功能至关重要。尽管生物物理技术为研究各种共溶剂存在下的酶的稳定性和活性提供了强大的工具，但结合实验技术和分子动力学（MD）模拟的方法可能会深入了解控制酶稳定性的相互作用机制。这些相互作用的知识可进一步用于对野生型酶进行各种修饰，以用于各种药物应用。在此，我们采用荧光、紫外-可见、圆二色性（CD）、动态光散射（DLS）研究和 MD 模拟，全面了解存在甜菜碱、肌氨酸、精氨酸和脯氨酸时的菠萝蛋白酶（BM）。发现 BM 在 1 M 渗透压调节剂存在下的热稳定性顺序为：脯氨酸＞甜菜碱＞缓冲液＞精氨酸＞肌氨酸。BM 在存在甜菜碱和脯氨酸时比在肌氨酸和精氨酸时更优先水合。然而，MD 模拟表明，除了脯氨酸外，1 M 的甜菜碱、肌氨酸和精氨酸通过氢键与 BM 的活性位点相互作用，脯氨酸负责更破坏活性位点。在 1 M 下，BM 的催化位点残基之间的距离分别为 1.6、1.9、4.3、5.0 和 6.2 Å，用于脯氨酸、缓冲液、甜菜碱、精氨酸和肌氨酸。据我们所知，这是首次详细明确报道在甲基胺和氨基酸存在下 BM 的变性和失活。
