A scaffolding protein is a multivalent hub for organizing bacterial cytoplasm

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Despite being the simplest organisms on our planet, bacteria exemplify a universal feature of life: they are exquisitely organized. Amazingly, the mixture of many thousands of different proteins in a bacterial cell is a self-organizing system. This is accomplished by the production of molecular scaffolds, which are large, three-dimensional, porous networks that attach to specific target proteins and group them into cooperative networks. The placement of these scaffolds at defined locations within the cell provides a basic form of bacterial anatomy. Although bacterial scaffolds have major influences on cell physiology, relatively little is known about their structure and function, and a major goal of this project is to understand scaffolds and their interactions with target proteins at a molecular level. A second goal is to understand how scaffold networks are different among related species that exhibit large variations in cell anatomy. This will provide novel insight on how the plasticity of scaffold networks facilitates the evolution of new bacterial species. The concept of exquisite organization within cells relates directly to the broader impacts of the project, which will come from the construction of a visually stunning interactive microscope exhibit that will be viewed by thousands of young children at the Children's Museum of Cheyenne, Wyoming. Many bacteria become organized by assembling polymeric protein scaffolds that recruit groups of regulatory proteins into cooperative networks. Although these scaffolds have major influences on bacterial physiology and cell organization, relatively little is known about their structure and function. This project addresses this significant knowledge gap with the following specific aims: (i) Identify the direct binding partners of a scaffolding protein, called PopZ, in the species Caulobacter crescentus, and characterize the binding interfaces at a molecular level. This will be the first comprehensive study of a bacterial scaffold interface, with mutational analyses that will provide a precise level of detail. (ii) Understand how the three-dimensional structure of the PopZ scaffold influences network assembly and the kinetic properties of the PopZ-binding proteins moving within it. This will be the first study to relate the three-dimensional geometry of the scaffold to its function within the cell, and will include novel mathematical models of particle movements based on empirically determined biophysical parameters. (iii) Connect changes in the PopZ amino acid sequence to changes in cell organization during the evolution of new species. This will be the first study to compare PopZ networks between species that have large differences in sub-cellular organization, and will test the hypothesis that scaffold networks provide a relatively promiscuous binding surface that facilitates evolutionary change. This project also emphasizes broader impacts that extend beyond basic science. In creating a unique and interactive live cell microscopy exhibit at the Children's Museum of Cheyenne, the investigators will inspire future generations of scientists by impressing them with the inherent beauty of sub-cellular organization.