Masnoddin Makdi, Ling Clemente Michael Wong Vui, Yusof Nur Athirah
Biotechnology Research Institute, Universiti Malaysia Sabah, Jalan UMS, Kota Kinabalu 88400, Sabah, Malaysia.
Preparatory Centre for Science and Technology, Universiti Malaysia Sabah, Jalan UMS, Kota Kinabalu 88400, Sabah, Malaysia.
Microorganisms. 2022 Aug 16;10(8):1654. doi: 10.3390/microorganisms10081654.
BG5 is an obligate psychrophilic bacterium that was first isolated on King George Island, Antarctica. Over the last 50 years, the West Antarctic, including King George Island, has been one of the most rapidly warming places on Earth, hence making it an excellent area to measure the resilience of living species in warmed areas exposed to the constantly changing environment due to climate change. This bacterium encodes a genome of approximately 5694 protein-coding genes. However, 35% of the gene models for this species are found to be hypothetical proteins (HP). In this study, three conserved HP genes of , designated , and , were cloned and the proteins were expressed, purified and their functions and structures were evaluated. Real-time quantitative PCR analysis revealed that these genes were expressed constitutively, suggesting a potentially important role where the expression of these genes under an almost constant demand might have some regulatory functions in thermal stress tolerance. Functional analysis showed that these proteins maintained their activities at low and moderate temperatures. Meanwhile, a low citrate synthase aggregation at 43 °C in the presence of PCBG5HP1 suggested the characteristics of chaperone activity. Furthermore, our comparative structural analysis demonstrated that the HPs exhibited cold-adapted traits, most notably increased flexibility in their 3D structures compared to their counterparts. Concurrently, the presence of a disulphide bridge and aromatic clusters was attributed to PCBG5HP1's unusual protein stability and chaperone activity. Thus, this suggested that the HPs examined in this study acquired strategies to maintain a balance between molecular stability and structural flexibility. Conclusively, this study has established the structure-function relationships of the HPs produced by and provided crucial experimental evidence indicating their importance in thermal stress response.
BG5是一种专性嗜冷细菌，最初是在南极洲的乔治王岛分离出来的。在过去的50年里，包括乔治王岛在内的南极西部一直是地球上变暖最快的地区之一，因此使其成为测量生活在因气候变化而不断变化的环境中的温暖地区的生物物种恢复力的绝佳区域。这种细菌编码一个约有5694个蛋白质编码基因的基因组。然而，发现该物种35%的基因模型是假设蛋白（HP）。在本研究中，克隆了BG5的三个保守HP基因，分别命名为PCBG5HP1、PCBG5HP2和PCBG5HP3，并对这些蛋白进行了表达、纯化及功能和结构评估。实时定量PCR分析表明这些基因是组成型表达的，这表明在几乎恒定的需求下这些基因的表达可能在热应激耐受性方面具有一些调节功能，发挥着潜在的重要作用。功能分析表明这些蛋白在低温和中温下保持其活性。同时，在PCBG5HP1存在的情况下，柠檬酸合酶在43℃时的低聚集表明了伴侣活性的特征。此外，我们的比较结构分析表明，这些HP表现出冷适应特性，最显著的是与同类相比其三维结构具有更大的灵活性。同时，二硫键和芳香簇的存在归因于PCBG5HP1不同寻常地蛋白质稳定性和伴侣活性。因此，这表明本研究中检测的HP获得了在分子稳定性和结构灵活性之间保持平衡的策略。总之，本研究建立了BG5产生的HP的结构-功能关系，并提供了关键的实验证据，表明它们在热应激反应中的重要性。
