Membrane protein complexes remain the last major challenge in biochemistry and molecular biology. Although major advances have been made crystallizing selected membrane proteins and complexes, many membrane proteins of unknown function remain to be studied. This is even truer for complexes in the membrane. Here we propose to tackle this problem by an integrated analysis of membrane protein complexes in E. coli, not only a major microbial model system but also the only species in which the topology of all membrane proteins has been determined experimentally. In the course of this project, we will purify most or all membrane protein complexes of E. coli and analyze their composition by mass spectrometry. We have successfully carried out such an analysis in yeast and thus predict to obtain several hundred protein complexes. Independently, we will screen all cytoplasmic domains of E. coli membrane proteins for interactions using multiple yeast two-hybrid systems. In addition, we will map interactions of membrane proteins using a bacterial two-hybrid system to ensure in vivo assay conditions. These studies will allow us to map the topologies of membrane protein complexes and link those proteins to soluble proteins in the cytoplasm. Finally, we will focus on uncharacterized membrane proteins found in the aforementioned screens, especially transporters, and analyze their in vivo interactions and functions in detail using mutations and specialized assays to determine their activities (such as transport) using high throughput protocols. Bioinformatic analysis will assist all of the three subprojects. To our knowledge no such integrated study of this scale has been attempted before. The results of this project will have a broad impact on membrane protein biology in both microbes and eukaryotes, including biotechnological and medical applications. For instance, membrane proteins are common targets of antimicrobials and this project will identify new targets but also elucidate the moleculr function of many of these (hitherto uncharacterized) transmembrane proteins.
Escherichia coli is an important model microbe and pathogen. Membrane proteins are critical for its survival, adaptation to environmental change, and its physiology such as nutrient uptake. This study aims at the systematic dissection of membrane protein complexes, their interactions and molecular functions in vivo, using mass spectrometry, interaction assays, and transporter activity assays. EDITORS COMMENTS
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