R-bodies are self-assembling protein polymers from bacterial ensymbionts of 'killer' Paramecium strains that confer to the host the ability to kill competing strains. The tightly coiled sub-micron sized R-body undergoes a massive pH induced conformational change into elongated rods tens of microns long, generating forces sufficient to rupture biological membranes. Purified recombinant R-bodies can undergo many successive rounds of piston-like extension and contraction using only the chemical energy from pH change. These robust nanomachines have enormous potential for biotechnological and therapeutic applications. Their ability to behave as sensitive switches and to do work on biological structures can be adapted for many functions - they have, for example, been proposed as systems for phagosomal delivery of bioactive molecules in a way that would mimic their biological function. The ability to tune R-body behavior was recently demonstrated by a panel of point mutants that alter the triggering pH for extension. However, the lack of detailed molecular understanding of their assembly and the mechanism of the pH-sensitive piston-like extension are major roadblocks to developing R-bodies for novel applications. This proposal aims to determine the molecular architecture of R-bodies using a hybrid structural approach, and to measure their biophysical properties using optical trapping force measurements. Visualizing the three-dimensional structure of R-bodies in coiled and extended states using cryo-EM will allow us to define the molecular mechanism of their piston-like behavior, laying the groundwork for tuning the structures to new functions. This request for administrative equipment supplement is to upgrade our Titan Krios cryo-electron microscope with ?fringe-free? illumination, a proven strategy for increasing the throughput of cryo-EM data collection, which will allow us to collect larger datasets faster, greatly expanding the capacity of our existing instrumentation.

Public Health Relevance

R-bodies are self-assembling protein-based molecular pistons that undergo many rounds of extension and contraction, used by some bacteria as weapons to puncture host cells. This award aims to visualize the three- dimensional architecture of R-bodies, to define how they extend and contract, and to measure the forces that they can generate. R-bodies have tremendous potential as biosensors and therapeutic delivery devices, and these studies will lay the groundwork for adapting these nanomachines to novel functions.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM127648-03S1
Application #
10132688
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Flicker, Paula F
Project Start
2018-06-01
Project End
2022-05-31
Budget Start
2020-06-01
Budget End
2021-05-31
Support Year
3
Fiscal Year
2020
Total Cost
Indirect Cost
Name
University of Washington
Department
Biochemistry
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195