Second order rate constants of donor-strand exchange reveal individual amino acid residues important in determining the subunit specificity of pilus biogenesis.
Second order rate constants of donor-strand exchange reveal individual amino acid residues important in determining the subunit specificity of pilus biogenesis.
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DOI：
10.1007/s13361-011-0146-4
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发表时间：
2011-07
影响因子：
3.2
通讯作者：
Ashcroft, Alison E.
中科院分区：
文献类型：
--
作者：
Leney, Aneika C.;Phan, Gilles;Allen, William;Verger, Denis;Waksman, Gabriel;Radford, Sheena E.;Ashcroft, Alison E.
P皮利是一种毛发状的粘附结构，通过分子伴侣-引导蛋白途径组装在致尿路感染大肠杆菌的外膜上。在这一途径中，分子伴侣-亚基复合物在周质中形成，并靶向OM组装平台，引导器。菌毛亚基显示由缺失的β-链引起的大沟，在分子伴侣-亚基复合物中，β-链由分子伴侣提供。在引导器处，菌毛亚基以称为“供体链交换（DSE）”的机制组装，由此由伴侣蛋白提供的β链被进入的亚基的N末端延伸（Nte）交换。这以拉链进拉链出的方式发生，从Nte中的限定残基P5开始，插入到凹槽中的限定位点，即P5口袋。在这里，电喷雾电离质谱法（ESI-MS）已被用来测量DSE率在体外。分子伴侣-亚基复合物和一系列在不同残基处取代的Nte肽之间的二级速率常数证实了Nte的P5残基在确定DSE速率中的重要性。此外，P5残基两侧的残基（P5 + 1和P5 - 1），其侧链远离亚基沟，也调节DSE的速率，最有可能是通过在DSE之前帮助Nte对接到接受亚基上的P5口袋中。开发的ESI-MS方法适用于测量DSE在菌毛生物发生率一般，并证明了ESI-MS的范围，在确定生物分子过程中的分子细节。本文的在线版本（doi：10.1007/s13361-011-0146-4）包含补充材料，可供授权用户使用。
P pili are hair-like adhesive structures that are assembled on the outer membrane (OM) of uropathogenic Escherichia coli by the chaperone-usher pathway. In this pathway, chaperone-subunit complexes are formed in the periplasm and targeted to an OM assembly platform, the usher. Pilus subunits display a large groove caused by a missing β-strand which, in the chaperone-subunit complex, is provided by the chaperone. At the usher, pilus subunits are assembled in a mechanism termed “donor-strand exchange (DSE)” whereby the β-strand provided by the chaperone is exchanged by the incoming subunit’s N-terminal extension (Nte). This occurs in a zip-in-zip-out fashion, starting with a defined residue, P5, in the Nte inserting into a defined site in the groove, the P5 pocket. Here, electrospray ionization-mass spectrometry (ESI-MS) has been used to measure DSE rates in vitro. Second order rate constants between the chaperone-subunit complex and a range of Nte peptides substituted at different residues confirmed the importance of the P5 residue of the Nte in determining the rate of DSE. In addition, residues either side of the P5 residue (P5 + 1 and P5 – 1), the side-chains of which are directed away from the subunit groove, also modulate the rates of DSE, most likely by aiding the docking of the Nte into the P5 pocket on the accepting subunit prior to DSE. The ESI-MS approach developed is applicable to the measurement of rates of DSE in pilus biogenesis in general and demonstrates the scope of ESI-MS in determining biomolecular processes in molecular detail. The online version of this article (doi:10.1007/s13361-011-0146-4) contains supplementary material, which is available to authorized users.
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影响因子：
64.5
作者：
Sauer, FG;Pinkner, JS;Hultgren, SJ
通讯作者：
Hultgren, SJ
影响因子：
3.6
作者：
Li, Qinyuan;Ng, Tony W.;Thanassi, David G.
通讯作者：
Thanassi, David G.
影响因子：
3.2
作者：
Ng, TW;Akman, L;Thanassi, DG
通讯作者：
Thanassi, DG
DOI：
10.1073/pnas.90.8.3670
发表时间：
1993-04-15
影响因子：
11.1
作者：
DODSON, KW;JACOBDUBUISSON, F;HULTGREN, SJ
通讯作者：
HULTGREN, SJ
DOI：
10.1073/pnas.91.25.11889
发表时间：
1994-12-06
影响因子：
11.1
作者：
ROBERTS, JA;MARKLUND, BI;NORMARK, S
通讯作者：
NORMARK, S