Probing cellular protein complexes using single-molecule pull-down

Cellular processes result from dynamic interactions between biomolecules. The gold standard method for investigating interaction between biomolecules is the pull-down assay. We have extended the conventional pull-down assay to its ultimate limit: the analysis of single biomolecular complexes. We achieve this goal by performing the pull-downs directly on to the surface of microscope slides and visualizing the captured biomolecules at single-molecule resolution using total internal reflection fluorescence (TIRF) microscopy. We name this technology single-molecule pull-down or SiMPull. In one reification biotinylated antibody against the protein of interest is immobilized on a flow chamber. The flow chambers are passivated using a polymer coating such that the cellular components do not bind, and the immobilized antibody specifically captures the target protein. Cell lysates are diluted to obtain isolated molecules suitable for single-molecule imaging. The target molecules are fluorescently labeled either using a genetically encoded fluorescent protein tag or using antibodies, and imaged using a single-molecule TIRF microscope. Using SiMPull we are able to discriminate between multiple association states of a protein as well as determine the stoichiometry of interaction. This technology is widely applicable to an array of biological contexts, and is suitable to analysis of endogenous protein complexes from animal tissue. In particular, we have used SiMPull to investigate the architecture and assembly of mechanistic target of rapamycin complexes. The complexes captured from cell extracts on to our imaging chambers retain their functional activities: thus SiMPull can be used as a preparatory tool for single-molecule biochemical analysis on proteins that cannot be readily purified or reconstituted. Finally, we have extended this approach to the analysis of lipid-protein interactions.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biophysics & Computnl Biology

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2014

Author dc:creator

• Jain, Ankur

Contributors dc:contributor

• Ha, Taekjip
• Chen, Jie
• Gruebele, Martin
• Myong, Su-A
• Prasanth, Supriya G.

• single-molecule
• fluorescence microscopy
• protein interactions

Statement dc:rights

• Copyright 2013 Ankur Jain

Language dc:language

en

Handle dc:identifier

http://hdl.handle.net/2142/46887

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/46887