University of Washington,
Keywords: protein complex, pH-driven, conformational shift, in situ
Summary:Elucidating the pH-driven conformational shift of R-bodies, a force generator in bacteria, with in situ AFM Shuai Zhang,1,2,* Guangyang Cai,3 Jiajun Chen,4 Paul Ashby,4 Justin Kollman,3 Jim De Yoreo1,2 1 Materials Science and Engineering, University of Washington, Seattle, WA 2 Physical Sciences Division, Pacific Northwest National Laboratory, Richland, WA 3 Department of Biochemistry, University of Washington, Seattle, WA 4 Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, CA * email@example.com R-bodies are protein complexes, assembled by bacterial endosymbionts of ‘killer’ Paramecium strains,  capable of reversibly shifting the shapes, within seconds, between a contracted coil and an elongated helical ribbon in response to pH changes.  This shape-shift could generate sufficient force, rupturing biological membranes for releasing other small molecules across biological membranes. Recently, the purified R-bodies have been approved to undergo hundreds of reversible extension and contraction cycles with repeated pH shifts. In the meanwhile, the amplitude of the corresponding conformational change did not decay over cycles. Hence, R-bodies have attractive potential in bio-engineering as protein machines for drug delivery, tissue engineering, sensors/actuators, 3D electronic materials, etc.  Determining their assembly structures and exploring their physical model of conformation transition are essential to understand how protein molecules interact and organize into a macromolecular complex and generate force with responses to stimuli using simple chemical energy. In this presentation, we will share the most recent in situ AFM results of R-bodies’ structural transitions between pH 5 and pH 7 captured by high-resolution AFM with an integrated microfluidic system. The results visualize the hierarchical assembly of R-bodies and their structural basis for the reversible shape shift between different pH values. Combing the cryo-TEM tomography, we find out that the R-bodies fragments have two sub-layers, which have different pH responses in molecular packs. In addition, the pH-responsive vectors of the two sub-layers do not co-align with each other. The two conformation mismatches together lead to the differential expansion and contraction of the layers causing the coiling and uncoiling of R-bodies. Last, although the C-term truncation mutants assemble intact, coiled structures, none were able to transition to helical rods at low pH. This suggests that structural changes at the C-terminus of RebA and RebB are essential for the large-scale conformational change of the assembly. 1. Pond, F.R., et al., R-body-producing bacteria. Microbiological reviews, 1989. 53(1): p. 25-67. 2. Polka, J.K. and P.A. Silver, A Tunable Protein Piston That Breaks Membranes to Release Encapsulated Cargo. ACS Synthetic Biology, 2016. 5(4): p. 303-311. 3. Asakawa, H., et al., Spatial Distribution of Lipid Headgroups and Water Molecules at Membrane/Water Interfaces Visualized by Three-Dimensional Scanning Force Microscopy. ACS Nano, 2012. 6(10): p. 9013-9020.