Guo Yi, Mahajan Anjali, Yuan Chunhua, Joo Sang Hoon, Weghorst Christopher M, Tsai Ming-Daw, Li Junan
Ohio State Biochemistry Program, The Ohio State University, Columbus, Ohio 43210, USA.
Biochemistry. 2009 May 19;48(19):4050-62. doi: 10.1021/bi802247p.
Ankyrin repeat (AR) proteins are one of the most abundant repeat protein classes in nature, and they are involved in numerous physiological processes through mediating protein/protein interactions. The repetitive and modular architecture of these AR proteins may lead to biochemical and biophysical properties distinct from those of globular proteins. It has been demonstrated that like most globular proteins, AR proteins exhibit a two-state, cooperative transition in chemical- and heat-induced unfolding. However, the biophysical characteristics underlying such cooperative unfolding remain to be further investigated. In the present study, we evaluated the conformational stability of a group of cyclin-dependent kinase (CDK) 4-interacting AR proteins, P16, P18, IkappaBalpha, gankyrin, and their truncated mutants under different conditions, including the presence of denaturants, temperature, and pH. Our results showed that the first four N-terminal ARs are required to form a potent and stable CDK4 modulator. Moreover, in spite of their similarities in skeleton structure, CDK4 binding, and cooperative unfolding, P16, P18, IkappaBalpha, and gankyrin exhibited considerably different biophysical properties with regard to the conformational stability, and these differences mainly resulted from the discrepancies in the primary sequence of the relatively conserved AR motifs. Our results also demonstrated that these sequence discrepancies are able to influence the function of AR proteins to a certain extent. Overall, our results provide important insights into understanding the biophysical properties of AR proteins.
锚蛋白重复序列（AR）蛋白是自然界中最丰富的重复蛋白类别之一，它们通过介导蛋白质/蛋白质相互作用参与众多生理过程。这些AR蛋白的重复和模块化结构可能导致其生化和生物物理特性不同于球状蛋白。已经证明，与大多数球状蛋白一样，AR蛋白在化学诱导和热诱导的去折叠过程中表现出两态协同转变。然而，这种协同去折叠背后的生物物理特征仍有待进一步研究。在本研究中，我们评估了一组细胞周期蛋白依赖性激酶（CDK）4相互作用的AR蛋白、P16、P18、IkappaBalpha、巨锚蛋白及其截短突变体在不同条件下的构象稳定性，这些条件包括变性剂的存在、温度和pH值。我们的结果表明，前四个N端ARs是形成有效且稳定的CDK4调节剂所必需的。此外，尽管P16、P18、IkappaBalpha和巨锚蛋白在骨架结构、CDK4结合和协同去折叠方面具有相似性，但它们在构象稳定性方面表现出相当不同的生物物理特性，这些差异主要源于相对保守的AR基序一级序列的差异。我们的结果还表明，这些序列差异能够在一定程度上影响AR蛋白的功能。总体而言，我们的结果为理解AR蛋白的生物物理特性提供了重要见解。
