Watching Proteins Fold (or Misfold) in vivo with Mass Spectrometry

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Project Summary/Abstract Structural biology is exquisitely adept at providing us with high-resolution images of completed, properly- assembled proteins; however, we have extremely limited structural information about the `construction process' behind how these beautiful structures come to be. Because nascent proteins are time-dependent, heterogeneous, and intimately associated with the ribosome and other cellular machineries that act co- translationally, nascent proteins are beyond the grasp of existing structural and biophysical techniques, which require pure samples and are carried out in vitro. This blind-spot in our structural framework is troubling, because the nascent stage is when proteins are most likely to misfold or become mistargeted to an incorrect compartment – processes responsible for pathologies as diverse as cystic fibrosis, neurodegeneration, ageing, and cancer. Indeed, because most studies of folding – carried out by refolding a purified protein following denaturation – are so disconnected from how proteins fold in real life, they have contributed relatively little to the development of medicines to treat the many diseases caused by protein misfolding and misassembly. To address this gap, my research group is developing new tools and approaches to capture structural and kinetic information of protein folding intermediates as they are being synthesized in living cells. Specifically, we utilize crosslinking mass spectrometry (XL-MS) – an emerging technique in structural biology – in which spatial information is converted into covalent bonds via crosslinking agents. Distance restraints are inferred by sequencing the resulting crosslinked peptide fragments with mass spectrometry. These restraints are then used to computationally model a 3-D protein structure or dock one protein with respect to another. The innovations described in this proposal show how we are expanding the XL-MS toolkit to be able to capture transient structures of protein folding intermediates as they are being biosynthesized in cells. The technologies that we generate will be of immediate use to other scientists studying protein folding, translation, and quality control; more broadly, they will serve as a paradigm of how to perform biophysical experiments on biomolecules in their native cellular milieu. This paradigm shift will enable us to address a number of pressing questions of biomedical importance, specifically: (1) How do mutations in genetic diseases cause proteins to misfold? What is the structure of these misfolded states, and could drugs be designed to intervene at this stage? (2) What actually happens to proteomes as cells age? (3) As for amyloid- forming proteins, what are the structures and interactions of the soluble oligomeric forms in their cellular environment (which have so far eluded characterization by existing techniques), and what makes them cytotoxic?