Team:UCLA/Project/Protein Cages

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BACKGROUND

METHODOLOGY

RESULTS

BIOBRICKS

Protein Cages

Background

Abstract

The goal of our project is to modify a protein cage such that disassembly can be induced by thrombin protease. Our protein cage is a previously-created synthetic cage composed of 12 subunits that form a tetrahedron. By creating mutant protein cages with inserted thrombin cleavage sites, disassembly could be induced by treatment with thrombin protease. We designed 13 mutant protein cages and successfully cloned our top candidate based on selection criteria. We were then able to successfully express and affinity purify both the native protein cage and the mutant protein cage. However, complications with protein aggregation occurred, and upon assaying for protein cage assembly using dynamic light scattering, we found that our cages did not assemble at the expected size of 16nm. Future directions will be focused on improving expression conditions for proper cage assembly, as well as on functionalizing the interior/exterior.

Introduction

The seed project for next year involves the controlled disassembly of protein cages in response to thrombin protease. What is a protein cage? A protein cage is a type of supramolecular assembly, where multiple protein subunits interact non-covalently in order to form a higher-order structure. Classic examples of protein assemblies include viral capsids, ferritins, and clathrin coats(1). The cage that we are working with is a previously-designed synthetic protein cage(2–4) (Figure 1), created by one of our advisors, Todd Yeates. To make a subunit of the cage, two distinct, naturally oligomerizing proteins were selected—a trimer, bromoperoxidase, and a dimer, M1 matrix protein. Each were fused with a linker to form a 109.5 degree angle. This angle is crucial in that it allows 12 subunits to come together, in order to create a cage that has tetrahedral symmetry(2).

Figure 1: Structure of a designed protein cage. Geometric models illustrate the design principle of fusing two distinct, naturally oligomerizing proteins together in a specific geometry. Twelve subunits combine to form a cage with tetrahedral symmetry. Figure is adapted from Lai, Y.-T., Cascio, D. & Yeates, T. O. Structure of a 16-nm Cage Designed by Using Protein Oligomers. Science (80-. ). 336, 1129–1129 (2012).

The goal of our project is to modify this protein cage such that disassembly can be induced by thrombin protease. Proteases are enzymes that cleave at specific amino acid sequences; therefore, if our cage contained the sequence shown here, it can be disassembled by thrombin-induced cleavage. This would have potential applications such as controlled release of loaded drugs/imaging molecules from inside the cage. So why did we pick thrombin to work with? Thrombin was specifically chosen due to it being relatively well-studied, and because of its role in the coagulation cascade5, which contributes to heart disease and stroke the first and fifth leading causes of death in the United States, respectively(7).

Methodology and Preliminary Results

Design of Protein Cage Mutants Using previous crystallographic data, we visualized the protein cage using pymol (Figure 2). We then identified potential insertion sites based on two criteria:
1. The insertion had to be sterically accessible to protease binding
2. The insertion had to theoretically have minimal effects on initial protein cage assembly.
13 Protein Cage Mutants were designed and listed on a table in our notebook. Link to Notebook. We successfully cloned our top protein cage mutant (Mutant 10), based on the above criteria.

Figure 2: Protein Cage Visualization. The 12-subunit protein cage (left) is highlighted with each 3-subunit apex highlighted. A single apex (right) with the potential insertion sites highlighted in red. The protein cage was visualized using pymol. PDB ID: 3VDX.

Purification of Protein Cage
Since the protein cage was cloned to contain a C-terminal 6-histidine tag, affinity purification was used to isolate the native cage and the mutant protein cage. The purification of the native protein cage was done using SDS-PAGE (Figure 3).

Figure 3: Affinity purification of native protein cage. Whole cell lysates were subjected to nickel-histidine affinity purification.

Assay for Protein Cage Formation
We assayed for cage formation by measuring hydrodynamic radius using dynamic light-scattering (DLS) (Figure 4, Figure 5). DLS is based on the principle that fluctuations in the Brownian motion of suspended particles of distinct sizes scatter coherent light with fluctuating intensities(6). Analysis of these fluctuating intensities allows for the determination of particle velocity, and thus size(6). Unfortunately, protein aggregation was a complication, and the correct cage size of 16nm was not observed for either cage we tested.

Figure 4: Assay of Native Protein Cage Assembly. The purified fractions of affinity purified native protein cage was used to assay for protein cage assembly by measuring hydrodynamic radius (nm). The expected size of the tetrahedral cage is 16nm.

Figure 5: Assay of Mutant Protein Cage 10 Assembly. The purified fractions of affinity purified Mutant Protein Cage 10 was used to assay for protein cage assembly by measuring hydrodynamic radius (nm). The expected size of the tetrahedral cage is 16nm.

Future Directions
For future directions, the remaining mutants will be cloned, and the expression conditions will be optimized in order to obtain properly folded cages. This will be assayed using techniques such as circular dichroism and dynamic light scattering. Cages that fold properly will then be assayed for thrombin cleavage. At which point, it would be possible to load the interior of the cage with drug/imaging molecules for release, or functionalize the exterior of the cage for specific receptor interactions. Though the protein cage project is in a nascent phase, we hope to explore more possibilities that may shed light on using them for future applications in medicine.

Achievements

1. Used Pymol software to model our protein cage and design 13 different protein cage mutants
2. Cloned the top protein cage mutant based on our selection criteria
3. Verified expression and affinity purification the native protein cage using SDS-PAGE

List of Biobricks

• Protein Cage Mutant 10 BBa_K1763421

References

1. Goodsell, D. S. & Olson, A. J. Structural Symmetry and Protein Function. Annu. Rev. Biophys. Biomol. Struct. 105–53 (2000).

2. Padilla, J. E., Colovos, C. & Yeates, T. O. Nanohedra: using symmetry to design self assembling protein cages, layers, crystals, and filaments. Proc. Natl. Acad. Sci. U. S. A. 98, 2217–2221 (2001).

3. Lai, Y.-T., Cascio, D. & Yeates, T. O. Structure of a 16-nm Cage Designed by Using Protein Oligomers. Science (80-. ). 336, 1129–1129 (2012).

4. Lai, Y.-T. & Al, E. Structure and Flexibility of Nanoscale Protein Cages Designed by Symmetric Self-Assembly. J Am Chem Soc 135, 7738–7743 (2013).

5. Adams, R. L. C. & Bird, R. J. Review article : Coagulation cascade and therapeutics update : Relevance to nephrology . Part 1 : Overview of coagulation , thrombophilias and history of anticoagulants. 462–470 (2009). doi:10.1111/j.1440-1797.2009.01128.x

6. Arzen, D., Co-advisor, R. P. & Ljubljana, D. K. Dynamic light scattering and application to proteins in solutions. (2010).

7. http://www.cdc.gov/nchs/fastats/leading-causes-of-death.htm