Instituto de Bioquı́mica Vegetal y Fotosı́ntesis, CSIC- Universidad de Sevilla, Seville, Spain.
Biochemistry. 2011 Nov 15;50(45):9767-78. doi: 10.1021/bi2009272. Epub 2011 Oct 24.
In cyanobacteria, ammonium is incorporated into carbon skeletons by the sequential action of glutamine synthetase and glutamate synthase (GOGAT). The activity of Synechocystis sp. PCC 6803 glutamine synthetase type I (GS) is controlled by a post-transcriptional process involving protein-protein interactions with two inactivating factors: the 65-residue-long protein (IF7) and the 149-residue-long one (IF17). The sequence of the C terminus of IF17 is similar to IF7; IF7 is an intrinsically disordered protein (IDP). In this work, we study the structural propensities and affinity for GS of IF17 and a chimera protein, IF17N/IF7 (constructed by fusing the first 82 residues of IF17 with the whole IF7) by fluorescence, CD, and NMR. IF17 and IF17N/IF7 are IDPs with residual non-hydrogen-bonded structure, probably formed by α-helical, turn-like, and PPII conformations; several theoretical predictions support these experimental findings. IF17 seems to fold upon binding to GS, as suggested by CD thermal denaturations and steady-state far-UV spectra. The apparent affinity of IF17 for GS, as measured by fluorescence, is slightly smaller (K(D) 1 μM) than that measured for IF7 (0.3 μM). The K(D)s determined by CD are similar to those measured by fluorescence, but slightly larger, suggesting possible conformational rearrangements in the IFs and/or GS upon binding. Further, the results with IFN17/IF7 suggest that (i) binding of IF17 to the GS is modulated not only by its C-terminal region but also by its N-terminus and (ii) there are weakly structured (that is, "fuzzy") complexes in the ternary GS-IF system.
在蓝藻中，铵通过谷氨酰胺合成酶和谷氨酸合酶（GOGAT）的顺序作用掺入碳骨架中。集胞藻 PCC 6803 谷氨酰胺合成酶 I 型（GS）的活性受涉及与两种失活因子（IF7 和 IF17）蛋白-蛋白相互作用的转录后过程控制。IF17 的 C 端序列与 IF7 相似；IF7 是一种无规卷曲蛋白（IDP）。在这项工作中，我们通过荧光、CD 和 NMR 研究了 IF17 和嵌合蛋白 IF17N/IF7（通过将 IF17 的前 82 个残基与 IF7 的全长融合构建）与 GS 的结构倾向和亲和力。IF17 和 IF17N/IF7 是 IDP，具有残留的非氢键结构，可能由 α-螺旋、转角样和 PPII 构象形成；一些理论预测支持这些实验结果。如 CD 热变性和稳态远紫外光谱所建议的，IF17 似乎在与 GS 结合时发生折叠。通过荧光测量的 IF17 对 GS 的表观亲和力略小（K(D)~1 μM），而 IF7 的亲和力（约 0.3 μM）。通过 CD 确定的 K(D)与通过荧光测量的 K(D)相似，但略大，表明在结合时 IF 与 GS 可能发生构象重排。此外，IFN17/IF7 的结果表明，（i）IF17 与 GS 的结合不仅受其 C 端区域调节，还受其 N 端调节，（ii）在三元 GS-IF 系统中存在结构较弱的（即“模糊”）复合物。
