CAREER: Research and education on protein folding as an energy source at the bacterial outer membrane

Georgia Tech Research Corporation, Atlanta GA

Investigators

Abstract

All Gram-negative bacteria possess two distinct membranes surrounding them. These membranes contain numerous membrane proteins. In the inner membrane, most proteins adopt a helical form termed alpha-helical fold, whereas in the outer membrane they almost all form sheet-like structures by side-by-side alignment of protein strands, which is termed beta-sheet fold. The outer membrane contains proteins with large, open pores, rendering it unable to contain chemical energy stored in small molecules such as ATP or in the form of an ion gradient. Therefore, an alternative source of energy is required for complex processes such as insertion of proteins to membrane or secretion of proteins through the membrane. It is the goal of this project to determine the feasibility of protein folding itself as the source of energy. This project seeks to change a common paradigm in high school biology instruction, in which cells and the systems contained within are often seen as static and highly directed. Through molecular visualization of simulation data, the dynamic nature of proteins becomes readily apparent. Multiple modules on specific topics, such as the assembly of membranes and diffusion through membrane-bound channels, will be developed and implemented in high school classrooms in the North Georgia area. These modules emphasize the construction of explanatory models for the observed behavior, a key element of future science standards. Their implementation will be aided by the development of a simplified interface for the visualization program VMD. Assessment of the modules' impact will first be conducted in three classrooms before expanding to a wider audience through web distribution. Graduate and undergraduate students will be highly involved in the entire process, giving them training beyond just research. Multiple theoretical models have been proposed to explain how the folding of proteins to form beta-sheet can provide energy. In this proposal, computational investigations will be performed to determine, the energetic aspects of the formation of these structures through protein folding (1) in isolation, (2) in the insertion of outer-membrane-bound proteins and (3) in the secretion of auto transporter domains across the membrane. Quantitative determination of the energetic aspects of these processes will be used to evaluate and refine existing models. Investigations into two specific systems will be used to elucidate how outer-membrane proteins are inserted by BamA and how virulence protein domains are secreted by autotransporters, both being energized by their own folding. The specific objectives are to (1) characterize the free-energy landscape and stability of beta-sheet structures; (2) determine how BamA catalyzes the insertion and folding of outer-membrane proteins; (3) resolve the molecular sequence of events in autotransporter folding; and (4) integrate dynamics of biological systems into high school instruction. The primary method to be used is molecular dynamics (MD) simulation. All simulation results will be closely coupled to experiments, both retrospectively and prospectively.