Self-assembled compartments are a fundamental feature of biology and understanding how smaller biological molecules self-assemble into large-scale structures is integral to understanding life’s origins. Nature has many examples of proteins that self-assemble into highly-symmetrical, closed, cage-like assemblies that support critical cellular functions such as metal storage, protein refolding, or the confinement of unstable reaction intermediates. There is great interest in repurposing these structures for application in nano-materials and nano-medicine such as drug delivery, vaccine development, imaging, and multi-enzyme cascade reactions. In this context, the design of novel self-assembling protein systems presents a challenging goal in synthetic biology that would allow for a wide range of enzymes and proteins to be used for the above applications.

In Nature individual protein subunits often assembly into large-scale structures that are strongly guided by symmetry. Depending on how the symmetry elements are arrayed, this can lead to structures that repetitively extend in one or two dimensions or assemble into closed geometric cages. Our goal is to develop a generalizable approach to assembling protein cages that is easily adaptable to almost any enzyme or protein that would be useful in a nanocage design. We utilize de novo designed coiled-coil proteins as small, simple symmetry elements. By fusing coiled coil domains of appropriate symmetry to a C3 symmetric trimeric protein we have successfully constructed tetrahedral, octahedral and icosahedral cages from a single protein building block. Our lab is now focused on generalizing this approach to assemble a wide range of proteins and exploring different applications of these designed nanocages.

Current Research Interests

• Formation of nanocages from dimeric, tetrameric, or pentameric building block proteins.

• Formation of nanocages from monomeric building block proteins via two unique coiled coil sections.

• Studying cage assembly with a multitude of biophysical techniques.

Icosahedral Nanocage Design

By fusing a pentameric coiled coil to a trimeric esterase (which we previously used to create tetrahedral and octahedral nanocages) we successfully engineered an icosahedral cage. (our largest cage to date). This 60 subunit complex was characterized by atomic force microscopy, scanning tunneling electron microscopy and cryo-electron microscopy. Remarkably, this nanocage had greatly increased thermal and chemical stability compared to the initial trimeric esterase, but lost no enzymatic activity. This result exemplifies the modularity of our nanocage design that allows different cage geometries to be constructed simply by changing the small coiled-coil assembly domains.

Recent Publication

Cristie-David, A. S.; Chen, J.; Nowak, D. B.; Bondy, A. L.; Sun, K.; Park, S. I.; Banaszak Holl, M. M.; Su, M.; Marsh, E. N. G., "Coiled-Coil-Mediated Assembly of an Icosahedral Protein Cage with Extremely High Thermal and Chemical Stability." J. Am. Chem. Soc. 2019, 141, 9207-9216.

Elaborating a Designed Protein Nanocage with Additional Protein Domains to Improve Assembly Efficiency