462828 Self-Assembly of Protein Nano-Shapes Using Synthetic Coiled Coils

Thursday, November 17, 2016: 2:50 PM
Golden Gate 6 (Hilton San Francisco Union Square)
Won Min Park1, Mostafa Bedewy2, Karl K. Berggren2,3 and Amy E. Keating1,4, (1)Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, (2)Research Laboratory of Electronics, Massachusetts Institute of Technology, Cambridge, MA, (3)Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, MA, (4)Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA

Protein self-assembly is an emerging and powerful approach to create structures at the nanometer scale. Driven by protein-protein interactions, protein subunits can be designed to self-assemble into supramolecular nanostructures with desired dimension and geometry. Recently, computational methods have been used to design complex protein interfaces,1 while rational protein design approaches using simple protein motifs such as coiled coils have been successfully applied to design artificial structures different from naturally occurring protein folds.2 Coiled coils are simple, rod-shaped, superhelical protein motifs composed of two or more α-helical peptides. They self-assemble into oligomers stabilized by specific interactions between the α-helical peptide subunits. Because their sequence-to-structure relation is well characterized, coiled coils are potentially useful self-assembling modules for design of artificial nanostructures. For example, an approach of covalent linkage and folding of different coiled-coil segments has been used to create a protein tetrahedron which does not exist in nature.3 Furthermore, such a rational approach can be extended to design other artificial protein shapes using new sets of coiled coils. In this study, we demonstrate self-assembly of triangular protein nanostructures using synthetic heterodimeric coiled coils. We recently reported interaction profiles of computational designed hetero-associating coiled-coil peptides. Among them, sets of coiled-coiled pairs that make specific binding interactions over weak undesired crosstalk with others are available, and these orthogonal pairs can potentially serve as specific synthetic modules for protein self-assembly.In our design, orthogonal coiled coil pairs are covalently linked and self-assemble into a triangular shape. We investigated mixing and thermal annealing of these protein building blocks, and maximized the yield of self-assembled triangular protein structures by manipulating the annealing temperature pathway. The fraction of self-assembled proteins was purified from the mixture of annealed proteins, using size-exclusion chromatography. Atomic force microscopy revealed the triangular shape of self-assembled proteins, and their hydrodynamic diameter and the supercoiled folding were characterized using dynamic light scattering and circular dichroism, respectively. Our approach is simple and broadly applicable to design of other artificial protein nanostructures, which provide great potential in the area of protein materials development.

1. N. P. King, J. B. Bale, W. Sheffler, D. E. McNamara, S. Gonen, T. Gonen, T. O. Yeates and D. Baker, Nature, 2014, 510, 103-108.
2. H. Gradišar, S. Božič, T. Doles, D. Vengust, I. Hafner-Bratkovič, A. Mertelj, B. Webb, A. Šali, S. Klavžar and R. Jerala, Nat. Chem. Biol., 2013, 9, 362-366.
3. K. E. Thompson, C. J. Bashor, W. A. Lim and A. E. Keating, ACS Synth. Biol., 2012, 1, 118-129.

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