Daughdrill G W, Hanely L J, Dahlquist F W
Institute of Molecular Biology, University of Oregon, Eugene 97403, USA.
Biochemistry. 1998 Jan 27;37(4):1076-82. doi: 10.1021/bi971952t.
FlgM is the inhibitor of sigma 28, a transcription factor specific for the expression of bacterial flagella and chemotaxis genes. FlgM is also exported from the cytoplasm to the outside of the cell during the process of flagella filament assembly. In the absence of its targets, FlgM is a dynamic, mostly unfolded, molecule [Daughdrill, G. W., et al. (1997) Nat. Struct. Biol. 4(4), 285-291]. The NMR resonance assignments, dynamics, and average secondary structure of this mostly unfolded form of FlgM are reported here. Because of the dynamic behavior of FlgM, the deviation of C alpha chemical shifts from the random coil values was used to test for the presence of secondary structure [Wishart, D. S., and Sykes, B. D. (1994) Methods Enzymol. 239, 363-392]. This analysis shows two contiguous regions in the C-terminal half of FlgM with helical C alpha chemical shifts. These two regions, M60-G73 and A83-A90, contained less than 10 medium-range NOEs, and the 15N relaxation parameters suggest the helical structure is not rigid. However, the C alpha chemical shifts of M60-G73, A83-A90, and other residues in the C-terminal half of FlgM shift toward their canonical random coil values with the addition of a chemical denaturant. Along with the values of the order parameter, S2, this observation suggests the C-terminal half of FlgM exists in an equilibrium structural state that is nonrandom. The same analysis of the N-terminal half of FlgM suggests it more closely resembles a random coil in conditions with and without denaturant. It appears the C-terminal half of FlgM lacks sufficient intramolecular contacts to form stable secondary or tertiary structures. It is known this C-terminal region becomes rigidly held when FlgM binds sigma 28 (Daughdrill et al., 1997), and it is possible that binding stabilizes the helical structure. The potential evolutionary relationship between the inhibitory interaction of FlgM with sigma 28 and the autoinhibition observed in sigma 70 is discussed.
FlgM是σ28的抑制剂，σ28是一种对细菌鞭毛和趋化性基因表达具有特异性的转录因子。在鞭毛丝组装过程中，FlgM也会从细胞质输出到细胞外。在没有其靶标的情况下，FlgM是一个动态的、大多处于未折叠状态的分子[Daughdrill, G. W.,等人(1997年)《自然结构生物学》4(4)，285 - 291]。本文报道了这种大多处于未折叠状态的FlgM的核磁共振共振归属、动力学和平均二级结构。由于FlgM的动态行为，利用Cα化学位移相对于无规卷曲值的偏差来检测二级结构的存在[Wishart, D. S.,和Sykes, B. D.(1994年)《酶学方法》239，363 - 392]。该分析表明，FlgM C端一半区域有两个相邻区域具有螺旋状的Cα化学位移。这两个区域，M60 - G73和A83 - A90，包含少于10个中程核Overhauser效应(NOE)，并且15N弛豫参数表明螺旋结构不刚性。然而，随着化学变性剂的加入，FlgM C端一半区域中M60 - G73、A83 - A90以及其他残基的Cα化学位移向其标准无规卷曲值移动。连同序参数S2的值，这一观察结果表明FlgM的C端一半区域存在于一个非随机的平衡结构状态。对FlgM N端一半区域的相同分析表明，在有和没有变性剂的条件下，它更类似于无规卷曲。看来FlgM的C端一半区域缺乏足够的分子内接触来形成稳定的二级或三级结构。已知当FlgM与σ28结合时，这个C端区域会被牢固地固定住(Daughdrill等人，1997年)，并且有可能结合使螺旋结构稳定。文中还讨论了FlgM与σ28的抑制相互作用和在σ70中观察到的自抑制之间潜在的进化关系。
