Ramos Joao, Laux Valerie, Haertlein Michael, Boeri Erba Elisabetta, McAuley Katherine E, Forsyth V Trevor, Mossou Estelle, Larsen Sine, Langkilde Annette E
Life Sciences Group, Institut Laue-Langevin, 71 Avenue des Martyrs, 38000 Grenoble, France.
Partnership for Structural Biology (PSB), 71 Avenue des Martyrs, 38000 Grenoble, France.
IUCrJ. 2021 Mar 6;8(Pt 3):372-386. doi: 10.1107/S2052252521001299. eCollection 2021 May 1.
This structural and biophysical study exploited a method of perdeuterating hen egg-white lysozyme based on the expression of insoluble protein in followed by in-column chemical refolding. This allowed detailed comparisons with perdeuterated lysozyme produced in the yeast , as well as with unlabelled lysozyme. Both perdeuterated variants exhibit reduced thermal stability and enzymatic activity in comparison with hydrogenated lysozyme. The thermal stability of refolded perdeuterated lysozyme is 4.9°C lower than that of the perdeuterated variant expressed and secreted in yeast and 6.8°C lower than that of the hydrogenated protein. However, both perdeuterated variants exhibit a comparable activity. Atomic resolution X-ray crystallographic analyses show that the differences in thermal stability and enzymatic function are correlated with refolding and deuteration effects. The hydrogen/deuterium isotope effect causes a decrease in the stability and activity of the perdeuterated analogues; this is believed to occur through a combination of changes to hydrophobicity and protein dynamics. The lower level of thermal stability of the refolded perdeuterated lysozyme is caused by the unrestrained Asn103 peptide-plane flip during the unfolded state, leading to a significant increase in disorder of the Lys97-Gly104 region following subsequent refolding. An ancillary outcome of this study has been the development of an efficient and financially viable protocol that allows stable and active perdeuterated lysozyme to be more easily available for scientific applications.
这项结构与生物物理研究采用了一种基于在大肠杆菌中表达不溶性蛋白质然后进行柱内化学重折叠来对鸡蛋白溶菌酶进行全氘代的方法。这使得能够与在酵母中产生的全氘代溶菌酶以及未标记的溶菌酶进行详细比较。与氢化溶菌酶相比，两种全氘代变体均表现出热稳定性和酶活性降低。重折叠后的全氘代溶菌酶的热稳定性比在酵母中表达和分泌的全氘代变体低4.9°C，比氢化蛋白低6.8°C。然而，两种全氘代变体表现出相当的活性。原子分辨率X射线晶体学分析表明，热稳定性和酶功能的差异与重折叠和氘代效应相关。氢/氘同位素效应导致全氘代类似物的稳定性和活性降低；据信这是通过疏水性和蛋白质动力学变化的组合而发生的。重折叠后的全氘代溶菌酶较低的热稳定性水平是由未折叠状态下Asn103肽平面不受限制的翻转引起的，导致随后重折叠后Lys97 - Gly104区域的无序度显著增加。这项研究的一个附带成果是开发了一种高效且经济可行的方案，使得稳定且有活性的全氘代溶菌酶更易于用于科学应用。
