Protection by desiccation-tolerance proteins probed at the residue level
Protection by desiccation-tolerance proteins probed at the residue level
复制标题
DOI：
10.1002/pro.4231
复制
发表时间：
2021-11-24
期刊：
影响因子：
8
通讯作者：
Pielak, Gary J.
中科院分区：
文献类型：
--
作者：
Crilly, Candice J.;Brom, Julia A.;Pielak, Gary J.
来自生命各个领域的极端耐受性生物产生保护性的内在无序蛋白(IDPs)来响应干燥压力。在体外，这些国内流离失所者中的许多人比美国食品和药物管理局批准的辅料更好地保护酶免受脱水压力。然而，与大多数赋形剂一样，人们对其保护机制知之甚少。在这里，我们应用热重分析、差示扫描量热法和液体观察气体交换(LOVE)核磁共振技术来研究两种模型球状蛋白(葡萄球菌蛋白G[GB1]的B1结构域和糜蛋白酶抑制物2[CI2])对两种干燥耐受蛋白(来自迟滞的CAHS D和来自厌水生物的PvLEA4)以及无序和球状蛋白对照的保护作用。我们发现，所有蛋白质样品都保持了相似的水量和相似的玻璃化转变温度，这表明增强的保水性或玻璃化都不是起到保护作用的。LOVE核磁共振显示，IDPs比球形蛋白对照更好地防止脱水诱导的折叠，一般保护GB1和CI2的相同区域，并且保护GB1比CI2更好。这些观察表明，静电相互作用、电荷图案化和扩展的构象是保护的关键。将LOVE核磁共振进一步应用于更多的客户蛋白质和保护剂，将加深我们对脱水保护的理解，使脱水蛋白质的生产得以简化，扩大在医疗、生物技术和化学工业中的应用。
Extremotolerant organisms from all domains of life produce protective intrinsically disordered proteins (IDPs) in response to desiccation stress. In vitro, many of these IDPs protect enzymes from dehydration stress better than U.S. Food and Drug Administration-approved excipients. However, as with most excipients, their protective mechanism is poorly understood. Here, we apply thermogravimetric analysis, differential scanning calorimetry, and liquid-observed vapor exchange (LOVE) NMR to study the protection of two model globular proteins (the B1 domain of staphylococcal protein G [GB1] and chymotrypsin inhibitor 2 [CI2]) by two desiccation-tolerance proteins (CAHS D from tardigrades and PvLEA4 from an anhydrobiotic midge), as well as by disordered and globular protein controls. We find that all protein samples retain similar amounts of water and possess similar glass transition temperatures, suggesting that neither enhanced water retention nor vitrification is responsible for protection. LOVE NMR reveals that IDPs protect against dehydration-induced unfolding better than the globular protein control, generally protect the same regions of GB1 and CI2, and protect GB1 better than CI2. These observations suggest that electrostatic interactions, charge patterning, and expanded conformations are key to protection. Further application of LOVE NMR to additional client proteins and protectants will deepen our understanding of dehydration protection, enabling the streamlined production of dehydrated proteins for expanded use in the medical, biotechnology, and chemical industries.