Design of an in vitro assay to optimize assembly of nanoparticle-tagged nuclear import complexes

Maintaining protein function at the biological-inorganic interface is a critical challenge for bionanotechnology. Specifically, nanoparticle-protein conjugates must be designed to interact with binding partners with biologically-relevant thermodynamics. Towards developing a nanoparticle-tagging system that minimizes interference with normal protein function, here we design and begin development of an assay to assess complex formation between nanoparticle-immobilized proteins and soluble binding partners. Two chaperone proteins, importin-a and importin-3 mediate classical nuclear transport, an essential and highly conserved example of protein complex formation in eukaryotic cells. Together, these two proteins form a chaperone complex that recognizes a nuclear localization signal (NLS), which is a short peptide sequence. Here, we synthesize and purify a fluorescently-labeled importin-a and a positive control for complex formation, which consists of bovine albumin serum (BSA) covalently conjugated to a fluorophore and NLS. Using these two fluorescent molecules, we can perform Forster Resonance Energy Transfer (FRET) experiments to study the kinetics and thermodynamics of these protein interactions. The development of this system will be used in future tests with the NLS-conjugated fluorescent gold nanoparticles.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Mechanical Engineering.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2007

Author dc:creator

• Rosenbaum, Lara Elise

Advisor dc:contributor.advisor

• Kimberly Hamad-Schifferli.

• Mechanical Engineering.

Statement dc:rights

• M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/40471

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/40471