Kumar Niti, Shukla Swati, Kumar Sanjiv, Suryawanshi Anju, Chaudhry Uma, Ramachandran Srinivasan, Maiti Souvik
Proteomics and Structural Biology Unit, Institute of Genomics and Integrative Biology, Mall Road, Delhi 110 007, India.
Proteins. 2008 May 15;71(3):1123-33. doi: 10.1002/prot.21798.
Compared to eukaryotes, the occurrence of "intrinsically disordered" or "natively unfolded" proteins in prokaryotes has not been explored extensively. Here, we report the occurrence of an intrinsically disordered protein from the mesophilic human pathogen Mycobacterium tuberculosis. The Histidine-tagged recombinant Rv3221c biotin-binding protein is intrinsically disordered at ambient and physiological growth temperatures as revealed by circular dichroism and Fourier transform infrared (FTIR) spectroscopic studies. However, an increase in temperature induces a transition from disordered to structured state with a folding temperature of approximately 53 degrees C. Addition of a structure inducing solvent trifluoroethanol (TFE) causes the protein to fold at lower temperatures suggesting that TFE fosters hydrophobic interactions, which drives protein folding. Differential Scanning Calorimetry studies revealed that folding is endothermic and the transition from a disordered to structured state is continuous (higher-order), implying existence of intermediates during folding process. Secondary structure analysis revealed that the protein has propensity to form beta-sheets. This is in conformity with FTIR spectrum that showed an absorption peak at wave number of 1636 cm(-1), indicative of disordered beta-sheet conformation in the native state. These data suggest that although Rv3221c may be disordered under ambient or optimal growth temperature conditions, it has the potential to fold into ordered structure at high temperature driven by increased hydrophobic interactions. In contrast to the generally known behavior of other intrinsically disordered proteins folding at high temperature, Rv3221c does not appear to oligomerize or aggregate as revealed through numerous experiments including Congo red binding, Thioflavin T-binding, turbidity measurements, and examining molar ellipticity as a function of protein concentration. The amino acid composition of Rv3221c reveals that it has 24% charged and 54.9% hydrophobic amino acid residues. In this respect, this protein, although belonging to the class of intrinsically disordered proteins, has distinct features. The intrinsically disordered state and the biotin-binding feature of this protein suggest that it may participate in many biochemical processes requiring biotin as a cofactor and adopt suitable conformations upon binding other folded targets.
与真核生物相比，原核生物中“内在无序”或“天然未折叠”蛋白质的存在尚未得到广泛研究。在此，我们报告了一种来自嗜温人类病原体结核分枝杆菌的内在无序蛋白质的存在。通过圆二色性和傅里叶变换红外（FTIR）光谱研究表明，带有组氨酸标签的重组Rv3221c生物素结合蛋白在环境温度和生理生长温度下是内在无序的。然而，温度升高会诱导其从无序状态转变为有序状态，折叠温度约为53摄氏度。添加结构诱导溶剂三氟乙醇（TFE）会使该蛋白在较低温度下折叠，这表明TFE促进了疏水相互作用，从而驱动蛋白质折叠。差示扫描量热法研究表明，折叠是吸热的，从无序状态到有序状态的转变是连续的（高阶的），这意味着在折叠过程中存在中间体。二级结构分析表明，该蛋白倾向于形成β-折叠。这与FTIR光谱一致，该光谱在波数1636 cm⁻¹处显示出一个吸收峰，表明在天然状态下存在无序的β-折叠构象。这些数据表明，尽管Rv3221c在环境温度或最佳生长温度条件下可能是无序的，但它有潜力在高温下通过增加的疏水相互作用驱动折叠成有序结构。与其他在高温下折叠的内在无序蛋白质的一般已知行为相反，通过包括刚果红结合、硫黄素T结合、浊度测量以及检查摩尔椭圆率作为蛋白质浓度函数的大量实验表明，Rv3221c似乎不会寡聚或聚集。Rv3221c的氨基酸组成显示其有24% 的带电荷氨基酸残基和54.9% 的疏水氨基酸残基。在这方面，这种蛋白质虽然属于内在无序蛋白质类别，但具有独特的特征。这种蛋白质的内在无序状态和生物素结合特性表明，它可能参与许多需要生物素作为辅因子的生化过程，并在与其他折叠靶标结合时采用合适的构象。
