FhuA interactions in a detergent-free nanodisc environment
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- 作者：Allan Mills ; Hai-Tuong Le ; James W. Coulton ; Franck Duong
- 关键词：BN ; blue-native ; CN ; clear-native ; DDM ; dodecyl-尾-d-maltoside ; LDAO ; N-lauryldimethyl amine-N-oxide ; Fc ; ferricrocin ; ITC ; isothermal titration calorimetry ; MSP ; membrane scaffold protein
- 刊名：Biochimica et Biophysica Acta (BBA)/Biomembranes
- 出版年：January, 2014
- 年：2014
- 卷：1838
- 期：1
- 页码：364-371
- 全文大小：1012 K
文摘
TonB-dependent membrane receptors from bacteria have been analyzed in detergent-containing solution, an environment that may influence the role of ligand in inducing downstream interactions. We report reconstitution of FhuA into a membrane mimetic: nanodiscs. In contrast to previous results in detergent, we show that binding of TonB to FhuA in nanodiscs depends strongly on ferricrocin. The stoichiometry of interaction is 1:1 and the binding constant KD is ~ 200 nM; an equilibrium affinity that is ten-fold lower than reported in detergent. FhuA in nanodiscs also forms a high-affinity binding site for colicin M (KD ~ 3.5 nM), while ferricrocin renders FhuA refractory to colicin binding. Together, these results demonstrate the importance of the ligand in regulating receptor interactions and the advantages of nanodiscs to study 尾-barrel membrane proteins in a membrane-like environment.