Elucidation of principles in the assembly of outer membrane proteins

通过外膜的呼吸电子转移是细菌电子传递链的一个关键特征，细菌电子传递链是向膜不渗透电子受体（如铁或锰氧化物）传递的。像铁（III）或锰（IV）这样的呼吸金属对全球碳循环有着深远的影响。在模式生物希瓦氏菌（Shewanella oneidensis）中，由质周细胞色素（MtrA）， β-桶蛋白（MtrB）和表面定位细胞色素（MtrC）组成的外膜复合物对催化异化铁和锰还原至关重要。这表明，为了允许MtrB的生产，MtrA必须存在。在这个研究项目中，我们的目标是阐明对MtrABC复合物形成的重要细胞功能，以及进行跨膜电子转移所需的结构先决条件。我们的初步工作表明，MtrA具有双重功能，既是一种电子转移蛋白，也是新生MtrB通过外周质运输所必需的支架或伴侣。通过交联和脉冲追踪实验，我们将检测MtrA和其他质周伴侣如何影响MtrB的传输，以及如何建立与MtrA的相互作用。我们将研究如何利用随机突变和交联来招募表面定位的细胞色素。此外，我们将尝试结晶MtrABC复合物并确定其分子结构。

Respiratory electron transfer over the outer membrane is a key feature within the bacterial electron transport chains to membrane impermeable electron acceptors like ferric iron or manganese oxides. Breathing metals like Fe(III) or Mn(IV) has a profound impact on the global carbon cycle. In the model organism Shewanella oneidensis an outer membrane complex consisting of a periplasmic cytochrome (MtrA), a β-barrel protein (MtrB) and a surface localized cytochrome (MtrC) is essential for the catalysis of dissimilatory ferric iron and manganese reduction. It was shown that MtrA has to be present in order to allow for the production of MtrB. In this research project we aim at elucidating cellular functions that are important for the formation of the MtrABC complex as well as the structural prerequisites necessary to conduct membrane spanning electron transfer. Our preliminary work suggests a dual function of MtrA, being an electron transferring protein and also a scaffold or chaperone necessary for the transport of nascent MtrB through the periplasm. Using cross-linking and pulse chase experiments we will detect how MtrA and other periplasmic chaperons influence MtrB transport and how the interaction with MtrA is established. We will investigate how the surface localized cytochrome is recruited by this complex using random-mutagenesis and cross-linking. In addition, we will attempt to crystallize the MtrABC complex and determine its molcular structure.