CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
Sci China Life Sci. 2011 Jun;54(6):513-9. doi: 10.1007/s11427-011-4182-x. Epub 2011 Jun 26.
Protein thermostability is an inherent characteristic of proteins from thermophilic microorganisms, and therefore enables these organisms to survive at extreme temperatures. Although it is well-known that thermostable proteins are critical for the growth of thermophilic organisms, the structural basis of protein thermostability is not yet fully understood. The histidine-containing phosphocarrier (HPr) protein, a phosphate shuttle protein in the phosphoenolpyruvate-dependent sugar transport system (PTS) of bacterial species, is an ideal model for investigating protein thermostability with respect to its small size and deficiency in disulphide bonds or cofactors. In this study, the HPr protein from Thermoanaerobacter tengcongensis (TtHPr) is cloned and purified. Crystal structure with good quality has been determined at 2.3 Å resolution, which provides a firm foundation for exploring the thermostable mechanism. However, it shows that the crystal structure is conserved and no clue can be obtained from this single structure. Furthermore, detailed comparison of sequence and structure with the homologs from meso- or thermophilic bacteria shows no obvious rule for thermostability, but the extra salt-bridge existing only in thermophilic bacteria might be a better explanation for thermostability of HPr. Thus, mutations are performed to interrupt the salt-bridge in HPrs in thermophilic bacteria. Using site-directed mutations and the circular dichroism method, thermostability is evaluated, and the mutational variations are shown to have a faster denaturing rate than for wild-type viruses, indicating that mutations cause instability in the HPrs. Understanding the higher-temperature resistance of thermophilic and hyperthermophilic proteins is essential to studies on protein folding and stability, and is critical in engineering efficient enzymes that can work at a high temperature.
蛋白质热稳定性是嗜热微生物蛋白质的固有特性，因此使这些生物能够在极端温度下生存。尽管众所周知，热稳定蛋白对于嗜热生物的生长至关重要，但蛋白质热稳定性的结构基础尚未完全了解。组氨酸含有磷酸载体（HPr）蛋白是细菌磷酸烯醇丙酮酸依赖型糖转运系统（PTS）中的磷酸穿梭蛋白，是研究蛋白质热稳定性的理想模型，因为其分子量小，缺乏二硫键或辅因子。在这项研究中，从 Thermoanaerobacter tengcongensis（TtHPr）克隆并纯化了 HPr 蛋白。已经以 2.3Å 的分辨率确定了高质量的晶体结构，为探索热稳定性机制提供了坚实的基础。然而，它表明晶体结构是保守的，从单个结构中无法获得任何线索。此外，与中温和嗜热细菌的同源物进行序列和结构的详细比较表明，没有明显的热稳定性规律，但仅存在于嗜热细菌中的额外盐桥可能是 HPr 热稳定性的更好解释。因此，对嗜热细菌中的 HPr 进行了突变以中断盐桥。通过定点突变和圆二色性方法评估热稳定性，突变显示出比野生型病毒更快的变性速率，表明突变导致 HPr 不稳定。了解嗜热和超嗜热蛋白的更高温度抗性对于研究蛋白质折叠和稳定性至关重要，并且对于工程能够在高温下工作的高效酶也至关重要。
