Design of an in vitro assay to optimize assembly of nanoparticle-tagged nuclear import complexes
Name
191748948-MIT.pdf
Description
Full printable version
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1.62 MB
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Checksum (MD5)
51e1f55adb77eeaf688bcd6433706e3d
Author(s)
Rosenbaum, Lara Elise
Advisor(s)
Kimberly Hamad-Schifferli.
Date Issued
2007
Publisher
Massachusetts Institute of Technology
Abstract
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.
Description
Thesis (S.B.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2007.
Includes bibliographical references (leaf 20).
Subjects
Mechanical Engineering.
MIT Department
Massachusetts Institute of Technology. Department of Mechanical Engineering
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