Signaling-Related Mobility Changes in Bacterial Chemotaxis Receptors Revealed by Solid-State NMR.
Signaling-Related Mobility Changes in Bacterial Chemotaxis Receptors Revealed by Solid-State NMR.
复制标题
DOI：
10.1021/acs.jpcb.7b06475
复制
发表时间：
2017-09-21
期刊：
影响因子：
--
通讯作者：
Thompson LK
中科院分区：
文献类型：
--
作者：
Kashefi M;Thompson LK
细菌使用非凡的膜结合纳米阵列来感知它们的环境并指导它们游泳。阵列由趋化受体二聚体三聚体组成，它们通过两种细胞质蛋白质环连接在膜的远端，一种是激酶CheA，另一种是偶联蛋白Chew。目前尚不清楚与受体周质结构域结合的配体如何使结合在细胞质尖端∼300away上的CHEA激酶失活，但其机制被认为涉及细胞质结构域内动力学的变化。为了验证这些建议，我们将固态核磁共振迁移率过滤实验应用于受体细胞质片段(U-13C，15N-CF)、CHEA和CHEW的功能复合体。这些蛋白质组装成天然的、均匀的阵列是由囊泡结合或分子拥挤剂介导的，顺磁松弛增强被用来克服这些大的复合体中的敏感性挑战。INEPT谱显示很大一部分受体在纳秒或更短的时间尺度上是动态的，这些动态随着信号状态的变化而变化。可移动区域通过生化和核磁共振方法(蛋白质截断和独特的化学位移)相结合来确定。INEPT谱与甲基化区域的不对称迁移率(N-螺旋迁移率、≫C-螺旋迁移率)相一致，并表明N-螺旋在去激活态的迁移率增加。这一发现确定了受体中功能相关的动力学，并表明这个N-螺旋片段在传播信号方面发挥了关键作用。
Bacteria employ remarkable membrane-bound nanoarrays to sense their environment and direct their swimming. Arrays consist of chemotaxis receptor trimers of dimers that are bridged at their membrane-distal tips by rings of two cytoplasmic proteins, a kinase CheA and a coupling protein CheW. It is not clear how ligand binding to the periplasmic domain of the receptor deactivates the CheA kinase bound to the cytoplasmic tip ∼300 Å away, but the mechanism is thought to involve changes in dynamics within the cytoplasmic domain. To test these proposals, we applied solid-state NMR mobility-filtered experiments to functional complexes of the receptor cytoplasmic fragment (U–13C,15N-CF), CheA, and CheW. Assembly of these proteins into native-like, homogeneous arrays is mediated by either vesicle binding or molecular crowding agents, and paramagnetic relaxation enhancement is used to overcome sensitivity challenges in these large complexes. INEPT spectra reveal that a significant fraction of the receptor is dynamic on the nanosecond or shorter time scale, and these dynamics change with signaling state. The mobile regions are identified through a combination of biochemical and NMR approaches (protein truncations and unique chemical shifts). The INEPT spectra are consistent with an asymmetric mobility in the methylation region (N-helix mobility ≫ C-helix mobility) and reveal an increase in the mobility of the N-helix in the kinase-off state. This finding identifies functionally relevant dynamics in the receptor, and suggests that this N-helix segment plays a key role in propagating the signal.
登录
查看更多内容
影响因子：
2.9
作者：
Briegel A;Wong ML;Hodges HL;Oikonomou CM;Piasta KN;Harris MJ;Fowler DJ;Thompson LK;Falke JJ;Kiessling LL;Jensen GJ
通讯作者：
Jensen GJ
DOI：
10.1073/pnas.1104824108
发表时间：
2011-06-07
影响因子：
11.1
作者：
Li, Mingshan;Hazelbauer, Gerald L.
通讯作者：
Hazelbauer, Gerald L.
影响因子：
8
作者：
Bartelli, Nicholas L.;Hazelbauer, Gerald L.
通讯作者：
Hazelbauer, Gerald L.
DOI：
10.1073/pnas.0802868105
发表时间：
2008-08-26
影响因子：
11.1
作者：
Besschetnova, Tatiana Y.;Montefusco, David J.;Weis, Robert M.
通讯作者：
Weis, Robert M.
影响因子：
2.9
作者：
Koshy, Seena S.;Li, Xuni;Thompson, Lynmarie K.
通讯作者：
Thompson, Lynmarie K.