Dynamics and equilibria of weak in-cell interactions

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In order to survive, cells must transmit signals, such as "attack this virus!" or build tiny structures, such as the spliceosome, a microscopic particle that cuts and splices the genetic code to get it ready to make proteins. All this involves proteins and nucleic acids folding and assembling inside the cell. In the past, this has been measured primarily in test tubes, but it really happens inside cells. This project will look inside single cells to measure accurately just how much folding and binding differ between cell and test tube. In addition to showing us how these molecular-level events happen in real life inside human cells, the project will train several PhD students and a postdoctoral fellow in modern techniques of molecular biophysics. It also supports training of undergraduate researchers to gain hands-on research skills long before the PhD stage. Lab members will participate in diversity-in-science outreach and meetings, to help the research enterprise reflect the diversity of US citizens.The proposed research goals, focusing on protein and RNA interactions inside living cells and organisms, will be achieved by combining new perturbation microscopy techniques (where temperature, osmotic pressure, and solvent conditions around cells are suddenly jumped) with large scale atomistic simulations of the interior of the cell (using supercomputers capable of simulating millions of atoms over hundreds of microseconds). The key systems to be investigated all revolve around biomolecule-biomolecule assembly (binding) and signaling. Assembly of tubulin protein dimers (part of the cell's skeleton) will be imaged inside cells, using a special tubulin variant that does not disrupt other tubulin activity in healthy cells. Assembly of viral capsids from hundreds of capsid proteins will be studied inside live cells. How does this assembly reaction manage to move backward when a virus infects, but forward when it is replicated and ready to leave the cell? Assembly of the U1A spliceosomal particle, which cuts pre-messenger RNA to make mRNA, will be monitored in its complex cycle both inside and outside of the nucleus. Assembly of heat shock chaperones with their clients and auxiliary factors to save proteins from misfolding will be compared in-cell and in the test tube. Folding of a two-domain protein inside a living vertebrate organism will be imaged for the first time. How do different tissues promote protein folding differently? Microsecond reaction-diffusion experiments in the whole cell at once will enable comparison with whole-cell models, as well as with molecular dynamics simulations of parts of the cytoplasm.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.