The structure and function of the beta-barrel assembly machinery
Lead Research Organisation:
University of Leeds
Department Name: Sch of Molecular & Cellular Biology
Abstract
The biogenesis of the periplasmic membrane is essential for viability and pathogenesis in Gram negative bacteria. The beta-barrel assembly machinery, or "BAM complex", is essential for this biogenesis, being required for the correct folding/insertion of outer membrane proteins (OMPS) into the bilayer of the periplasmic membrane. In E. coli, BAM is a complex of five proteins with a mass of ~203kDa, including an integral membrane, beta-barrel protein BamA, and four lipoproteins BamB-E. The mechanism by which BAM facilitates OMP folding/insertion is poorly understood, but understanding this mechanism may open exciting new avenues to tackle anti-microbial resistance.
Recent breakthroughs in cryo-EM and functional studies of BAM-assisted folding mean that we are perfectly positioned to address important questions in BAM structure and function for the first time
Objectives:
Combining cryo-EM with biochemical/biophysical assays we will:
1. Determine the atomic structure of the BAM complex. Building on exciting recent progress in the Ranson/Radford labs (Iadanza et al, Nature Communications., DOI:10.1038/ncomms12865), we will determine the atomic resolution structure of BAM using cryo-EM
2. Probe the role of conformational change in BAM function. We will use in vitro membrane protein folding assays and cryo-EM, and existing and new disulphide mutants that 'lock' BAM in different conformations.
3. Develop new methods for membrane protein structure determination. We will explore using SMA (styrene maleic acid) copolymers to extract BAM from its native membranes for structural biology.
Novelty/Timeliness:
The results will generate the first atomic solution structure of BAM, elucidate the importance of conformational change in BAM function, and lay the groundwork for the first structures of BAM in a lipid bilayer.
Experimental Approach:
We will utilize state of the art cryo-EM and biochemistry/biophysical techniques, offering the student training in a wide range of methodologies. The project is feasible, and built on strong preliminary data, ensuring success.
The project proposed sits squarely at the heart of the theme of the DTP proposal in "Mechanistic Biology and its Strategic Application" in that it
- embraces the study of basic, life-governing processes...from the atomic scale to the whole organism
- includes excellent molecular and cellular biosciences research
It also matches will with the BBSRC theme "World class Underpinning Bioscience" in that it will:
- Develop "highly skilled researchers"
- Ensure "strength in core underpinning disciplines such as cellular, molecular and structural biology"
- Drive "discovery of new leads of drugs or prevention strategies through improved understanding of biological mechanism"
- Build "new tools and technologies that enable researchers to push the boundaries".
Recent breakthroughs in cryo-EM and functional studies of BAM-assisted folding mean that we are perfectly positioned to address important questions in BAM structure and function for the first time
Objectives:
Combining cryo-EM with biochemical/biophysical assays we will:
1. Determine the atomic structure of the BAM complex. Building on exciting recent progress in the Ranson/Radford labs (Iadanza et al, Nature Communications., DOI:10.1038/ncomms12865), we will determine the atomic resolution structure of BAM using cryo-EM
2. Probe the role of conformational change in BAM function. We will use in vitro membrane protein folding assays and cryo-EM, and existing and new disulphide mutants that 'lock' BAM in different conformations.
3. Develop new methods for membrane protein structure determination. We will explore using SMA (styrene maleic acid) copolymers to extract BAM from its native membranes for structural biology.
Novelty/Timeliness:
The results will generate the first atomic solution structure of BAM, elucidate the importance of conformational change in BAM function, and lay the groundwork for the first structures of BAM in a lipid bilayer.
Experimental Approach:
We will utilize state of the art cryo-EM and biochemistry/biophysical techniques, offering the student training in a wide range of methodologies. The project is feasible, and built on strong preliminary data, ensuring success.
The project proposed sits squarely at the heart of the theme of the DTP proposal in "Mechanistic Biology and its Strategic Application" in that it
- embraces the study of basic, life-governing processes...from the atomic scale to the whole organism
- includes excellent molecular and cellular biosciences research
It also matches will with the BBSRC theme "World class Underpinning Bioscience" in that it will:
- Develop "highly skilled researchers"
- Ensure "strength in core underpinning disciplines such as cellular, molecular and structural biology"
- Drive "discovery of new leads of drugs or prevention strategies through improved understanding of biological mechanism"
- Build "new tools and technologies that enable researchers to push the boundaries".
