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Structural Studies and Engineering of Proteinaceous Assemblies

  • Author(s): McNamara, Dan Ethan
  • Advisor(s): Yeates, Todd O
  • et al.
Abstract

All aspects of life are controlled by proteins which often assemble into complex arrangements. Research on natural and unnatural protein assemblies enhances our understanding of biological science. The research described here provides structural and mechanistic insights into how protein assemblies could be utilized to improve or develop novel protein functions. Bacterial protein assemblies, microtubule-targeting compounds, and designed protein assemblies were investigated with X-ray crystallography and our results may further the understanding of these targets in pursuit of therapeutics for cancer, obesity, and other conditions.

The first study revealed an unexpectedly large structure and putative mechanism for the enzyme DmrB that may help in the design of anti-obesity drugs targeting gut bacteria. A second effort helped to explain how the outer shells of huge bacterial microcompartments may assemble together. The research carried out may help better implement efforts underway to engineer more stable microcompartments for new biotechnology approaches. Structural studies of the interactions which mediate enzyme encapsulation in bacterial microcompartments were also pursued. Our results establish methods for the isolation and subsequent characterization of binary and potentially ternary complexes of microcompartment shell proteins and luminal enzymes.

Current tubulin-targeting cancer therapeutics suffer from many drawbacks underscoring the need to further understand the structure of eukaryotic protein assemblies such as microtubules. We describe the structural characterization of two recently-identified compounds that potently kill cancer cell lines. High-throughput screening identified MI-181 and C2 in contexts of mitotic inhibition and cancer cell-death. X-ray crystal structures of MI-181 and C2 bound to a complex containing αβ-tubulin, tubulin tyrosine ligase, and the tubulin-binding protein stathmin revealed details of how these compounds interact with β-tubulin subunits. These structures clarify the unknown binding properties and may guide the improvement of these compounds as candidates for therapeutics or chemical probes for better understanding cell division.

In addition to natural protein assemblies, unnatural symmetric protein materials can be achieved through protein design. We have solved crystal structures of four computationally-designed protein assemblies. Additional structural studies of designed proteins revealed structures of sequences engineered to form cyclic oligomers and three-dimensional lattice symmetries. These structures emphasize the design accuracy that can be achieved using computational methods in favorable cases. These most recent designed assemblies are built from two distinct components, a strategy that lends itself to versatile construction of functional nanomaterials for a variety of applications.

Furthermore, we have developed an additional new method to design two-component protein assemblies which may be engineered for new functions. Fusion of a self-associating heterologous pair of helical elements to symmetric oligomers can be implemented in a way that generates three-dimensional closed systems or extended arrays of protein. Protein engineering applications are proposed and intial designs are described for several biotechnology purposes.

Self-assembling protein designs will contain short peptide ligands for cancer-related growth receptors. Designs in which symmetric oligomers self-assemble into extended rod-like filaments were tested for this application. Proper assembly was investigated with electron microscopy after purification from bacteria. The coiled-coil fusion design approach identified candidates for novel nanomaterials which may induce cellular responses related to cell growth and cancer in cultured endothelial cells.

Finally, improving the properties of key enzymes can overcome limitations of in vitro metabolic engineering systems. Enzymes for synthesizing isoprene – a starting compound for generating a variety of value-added products – are a useful test-bed. We incorporated a dimeric plant enzyme for isoprene synthesis into the design of a single-layer array of self-assembling symmetric oligomeric enzymes. Two designs are described which enable the isolation of two components separately which may self-assemble upon mixing to form a two-dimensional layer.

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