High-throughput analysis of protein dynamics

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Protein dynamics can be thought of as a chain of events that cellular proteins undergo from the moment of biosynthesis and until elimination. Examples of such events are synthesis on ribosomes, protein folding, interaction with other proteins, proteasomal degradation etc... In spite of the paramount importance, protein dynamics is a highly understudied topic. For example, the assembly pathways (the way proteins interact with one another to form complexes) are known only for a handful of ~4000 human protein complexes. This project focuses on the development of tools to explore dynamics of cellular protein complexes.

Our approach to tackle protein dynamics is based on the idea that biogenesis of protein complexes can be seen as a flow of metabolic reactions except that the "metabolites" are not typical small molecules but cellular proteins and their assemblies. Based on this idea we have recently developed an approach entitled kinetic analysis of incorporation rates in macromolecular assemblies (KARMA) that utilises a combination of isotope metabolic labelling and quantitative mass spectrometry to dissect in vivo dynamics of protein complexes. This project amis at development of the analytical techniques to address protein dynamics of various cellular protein complexes, host-pathogen interactions or other kinds of biomolecules.

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Cellular proteins form thousands of different complexes but almost nothing is known about how they assemble or change in time. To study these processes we have developed Kinetic Analysis of incorporation Rates in Macromolecular Assemblies (KARMA) - a method that relies on metabolic labelling to lean about the timing and order of the protein complexes assembly. In KARMA the complex assembly is treated akin to metabolic reactions, each taking some time. This creates a delay between the initial metabolite exposure (e.g. feeding cells with isotope labeled amino acids) and the time it incorporates into the complexes. This delay can be experimentally measured by isolating the protein complex through different complex components as affinity baits and analysing the metabolic label content by mass spectrometry. The complex constituent's labelling is then matched with a theoretical description - Kinetic State Model - to find the maturation time for every complex component, fraction of proteins in the assembly intermediates and and other important parameters. We apply this strategy to study various assembly processes.

Contributions

Project design: Evgeny Onishchenko (University of Bergen)
Biochemistry: Evgeny Onishchenko (University of Bergen)
Mass spectrometry: Ludovic Gillet (ETHZ/Picotti lab); Evgeny Onishchenko (University of Bergen)
Mathematical modelling: Elad Noor (Weizmann Institute/Milo lab)
Microscopy & genetics: Jonas Fischer (ETHZ/Weis lab)
Image analysis: Pascal Vallotton (Roche AG); Jonas Fischer (ETHZ/Weis lab)
Visualisation: Jonas Fisher (ETHZ/Weis lab); Matthias Wojtynek (ETHZ/Weis lab); Olga Posukh (Institute of Molecular and Cell Biology, Novosibirsk)
Funding and research environment: Karsten Weis (ETHZ); Evgeny Onishchenko (University of Bergen)