The Department of Mechanical Engineering in Partial Fulfillment of the Requirements for the Degree of Bachelor of Science at the Massachusetts Institute of Technology

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 nanoparticleimmobilized 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. Thesis Supervisor: Kimberly Hamad-Schifferli Title: Assistant Professor of Mechanical Engineering and Biological Engineering Introduction Nanoparticles have many unique properties that allow them to be used as fluorescent, magnetic, electron-density, or spectrophotometric tags on biomolecules. Using nanoparticles as tags offers biologists many new technological possibilities that are only starting to be developed. One method of particular interest is to use these nanoparticles to monitor protein complex formation. In this project, we have designed and developed a nanoparticle-tagging system to minimize interference with normal protein function. To do so, we have first designed an in vitro assay to study the proteins of interest under normal physiological conditions, as documented in the literature. In future studies, nanoparticles will be used to tag some of these proteins, and this novel approach can be used to provide highly quantitative data. 1. Nuclear Import One method employed by cells to transport proteins from the cytoplasm into the nucleus is through the use of members of the importin family, an essential and highly conserved example of protein complex formation in eukaryotic cells. This task can be broken down into two main steps: nuclear import complex formation followed by the nuclear import process. The two nuclear transport proteins, called importin-a and importin-P, form a chaperone complex which recognizes a nuclear localization signal (NLS), which is a short peptide sequence within a protein. When the importin-alimportin-0 complex binds to an NLS on a cargo protein, the entire chaperone-cargo complex is imported from the cytoplasm into the nucleus of the cell. This process is important for normal cellular function 2; therefore, it is a valuable test-bed on which to develop our technology. A. Nuclear Import Complex Formation Both importin-a and importin-3 are necessary for the nuclear import complex formation. Importin-a contains two NLS binding sites and an importin-p-binding (IBB) domain. An NLS binding site recognizes and attaches to an NLS in a protein. The IBB domain is an autoinhibitory domain which binds to the NLS binding sites in the absence of importin-3. Once importin-P binds to the IBB domain of importin-a, importin-a has a high affinity for and binds to an NLS sequence' (Figure 1). As there is only one IBB domain in importin-ca, importin-a and importin-0 form a complex in a 1:1 molar ratio. B. Nuclear Import Process Once the entire nuclear import complex has formed, it can be transported from the cytoplasm into the nucleus of the cell. Importin-0 is responsible for docking importin-a and its cargo (the protein containing the NLS) to the nuclear pore complex (NPC). Importin-P, importin-a, and the NLS-containing protein are then brought through the NPC and into the nucleus of the cell. There, by interacting with another protein, importin-P is removed from importin-a, releasing the IBB domain, and importin-a releases its cargo and converts to the lowaffinity form by binding to the freed IBB domain. Importin-a and importin-0 are then exported from the nucleus to be used again in the cytoplasm.2'3 (Figure 2) Figure 2. Diagram of Nuclear importation (Figure modified from (5) Lelyveld 2006). NLS = NLS-containing protein, a = importin-a, 13 = importin-1, RanGTp: This protein causes importin-13 to release the IBB domain, leading to the dissociation of the entire importin-a, importin-13, and NLS-containing protein complex.2 (A) (B) (C) NLS binding sites IBB domain Figure 1. Nuclear import protein complex formation (Figure modified from (5) Lelyveld 2006). a = importin-a, 3 = importin-j3, NLS = NLScontaining protein. (A) Importin-a, contains two NLS binding sites that are blocked by the IBB domain. (B) Importin-03 binds to the IBB domain of importin-a, exposing the two NLS binding sites. (C) An NLS conjugated to a nanoparticle binds to an NLS binding site on importin-a. 2. Forster Resonance Energy Transfer (FRET) Forster Resonance Energy Transfer (FRET) is a well-established technique for monitoring interactions between biomolecules. For the in vitro assay, a short NLS has been covalently attached to fluorescently-labeled bovine serum albumin (BSA), and importin-a has been labeled with fluorescein, a chemical fluorophore. By using Forster Resonance Energy Transfer (FRET) between the two fluorescent tags, the kinetics and thermodynamics of the protein interactions can be quantitatively measured and analyzed. The system has been designed such that the conditions are as close to physiological-relevance as possible. Each part of the assay has been optimized, including the stoichiometry of complex components and the reaction conditions for importin-a fluorescent labeling and BSA fluorescent labeling and NLS conjugation. Additionally, the proper design of the interface between the two proteins is critical. The dynamic protein structure is an important parameter to consider to prevent labeling the proteins in such a way that interferes with binding. FRET takes advantage of overlapping excitation and emission spectra of different fluorescent molecules or fluorophores. One fluorophore, the donor, can be excited, and the excited electrons within the fluorophore transfer their energy to a nearby different fluorophore, the acceptor. This different fluorophore then emits its own unique spectrum (Figure 3). There is no photon release by the donor fluorophore. The proximity of the two fluorophores is extremely important and occurs most efficiently at distances of 2 10 nm.7 This range works well for the vast majority of protein-protein interactions which take place over similar distances. Donation Excitation f Emission