Division of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences, Nanyang Technological University, Singapore.
J Biol Chem. 2010 Apr 16;285(16):12078-86. doi: 10.1074/jbc.M109.092445. Epub 2010 Feb 5.
The fundamental process of protein self-assembly is governed by protein-protein interactions between subunits, which combine to form structures that are often on the nano-scale. The nano-cage protein, bacterioferritin from Escherichia coli, a maxi-ferritin made up of 24 subunits, was chosen as the basis for an alanine-shaving mutagenesis study to discover key amino acid residues at symmetry-related protein-protein interfaces that control protein stability and self-assembly. By inspection of these interfaces and "virtual alanine scanning," nine mutants were designed, expressed, purified, and characterized using transmission electron microscopy, size exclusion chromatography, dynamic light scattering, native PAGE, and temperature-dependent CD. Many of the selected amino acids act as hot spot residues. Four of these (Arg-30, which is located at the two-fold axis, and Arg-61, Tyr-114, and Glu-128, which are located at the three-fold axis), when individually mutated to alanine, completely shut down detectable solution formation of 24-mer, favoring a cooperatively folded dimer, suggesting that they may be oligomerization "switch residues." Furthermore, two residues, Arg-30 and Arg-61, when changed to alanine form mutants that are more thermodynamically stable than the native protein. This investigation into the structure and energetics of this self-assembling nano-cage protein not only can act as a jumping off point for the eventual design of novel protein nano-structures but can also help to understand the role that structure plays on the function of this important class of proteins.
蛋白质自我组装的基本过程受亚基之间的蛋白质-蛋白质相互作用控制，这些相互作用结合形成通常处于纳米尺度的结构。纳米笼蛋白，来自大肠杆菌的菌铁蛋白，由 24 个亚基组成的大型铁蛋白，被选为丙氨酸削峰突变研究的基础，以发现控制蛋白质稳定性和自我组装的对称相关蛋白质-蛋白质界面的关键氨基酸残基。通过检查这些界面和“虚拟丙氨酸扫描”，设计了九个突变体，使用透射电子显微镜、尺寸排阻色谱、动态光散射、天然 PAGE 和温度依赖的 CD 进行表达、纯化和表征。选择的许多氨基酸充当热点残基。其中四个（位于二倍轴的 Arg-30 和位于三倍轴的 Arg-61、Tyr-114 和 Glu-128），当单独突变为丙氨酸时，完全阻止可检测的 24 聚体溶液形成，有利于协同折叠二聚体，表明它们可能是寡聚“开关残基”。此外，两个残基 Arg-30 和 Arg-61 突变为丙氨酸后形成的突变体比天然蛋白热力学更稳定。对这种自组装纳米笼蛋白的结构和能量学的研究不仅可以作为最终设计新型蛋白质纳米结构的起点，还可以帮助理解结构在这一类重要蛋白质的功能中的作用。
