Symmetry-Based Design of Protein Nano-Cages

[摘要] The self-assembly of protein subunits into large-scale oligomeric structures is a powerful and ubiquitous feature of biology, with viral capsids providing an excellent example. These assemblies perform a diverse set of functional and structural roles in living systems. Additionally, proteins can be modified both genetically and chemically to introduce new properties. Because of these attractive properties, natural and de novo designed protein assemblies have already been evaluated for various applications in medicine and materials science. This thesis explores a recently developed, generalizable coiled coil based strategy for de novo designing protein cages to utilize for applications in these fields.The design strategy relies on the combination of 2 rotational symmetry elements, one provided by the natural, building block protein (BBP) and the other provided by the coiled coil, to specify a protein cage of the desired geometry. The oligomerization of the coiled coil brings the copies of the BBP together, leading to the assembly of protein cages.By employing BBPs and coiled coils of different rotational symmetries, cages of various sizes and geometries have been designed, including both tetrahedral and octahedral protein cages. This thesis extends these studies to more ambitious design targets and explores the generalizability of this approach.Because the design strategy requires well-specified homo-oligomeric parallel-coiled coils, the utility of several selected de novo designed coiled coils was first evaluated as off-the-shelf components for protein assembly, using green fluorescent protein as a model system. This study revealed context-dependent oligomerization state changes for some of these coiled coils. Next, the potential of elaborating previously designed protein cages by attaching additional protein domains to free end of the coiled coil was investigated. As a proof-of-concept, an octahedral cage was elaborated by fusing a large monomeric protein to the free end of the coiled coil assembly domain. This design successfully self-assembled into a homogeneous octahedral protein cage of ~ 1.8 MDa, significantly the addition of the extra protein domain dramatically improved the yield and efficiency of protein assembly.The design strategy was extended to the de novo design of an icosahedral protein cage by fusing a pentameric coiled coil to the trimeric BBP previously utilized for octahedral and tetrahedral cage designs. After optimization, a construct with an 8-residue oligo-glycine spacer successfully assembled into a hyperstable 60-subunit protein cage with icosahedral geometry and molecular weight of ~ 2.1 MDa. Surprisingly, these cages captured short DNA strands during purification which were important to maintain the homogeneity of the cages. The cages could be transiently disassembled by treating with Dnase; the re-assembled cages were significantly more heterogeneous. The hyperstability and ability to capture DNA are new emergent properties of this design that arise from assembly and were not evident in previously designed cages. Finally, the potential of extending this symmetry-based strategy to design protein cages that assemble in response to environmental stimuli was investigated. This study was conducted by fusing a de novo designed metal-dependent coiled coil to the trimeric BBP. The construct successfully assembled into discrete particles in the presence of divalent transition metal ions; adding metal chelators or decreasing pH led to disassembly of these particles into their trimeric form.

[发布机构] University of Michigan

[效力级别] Protein design and engineering

[关键词] Protein Nano-cages;Protein design and engineering;Coiled-coil mediated protein assemblies;Biological Chemistry;Science;Chemistry