Rational and Computational Design of Superhelical Protein Nanotubes Open Access
Hughes, Spencer (Summer 2019)
Abstract
The design of peptide- and protein-based nanomaterials with high fidelity has long been a challenge in structural biology and materials science alike. The translation from the amino acid sequence to the folded and assembled structure is rarely facile, so design rules have been tabulated for the simplest of super-secondary structural elements (SSEs), such as coiled-coils and certain β-sheet assemblies. However, it has been shown that conservative mutations in these scaffolds has led to significant structural deviation. The search for a more predictable biomaterials scaffold has led to the characterization of Tandem Repeat Proteins (TRPs). The folding behavior of TRPs can be understood as a series of noncovalent lateral interactions between adjacent, nearly identical SSEs; in fact, the sequences of each SSE within a TRP can be aligned and statistically analyzed to derive consensus sequences for each TRP family. Consensus sequences serve as a mutagenesis guide; highly conserved positions should be held constant, whereas hypervariable positions are open for redesign. Using this strategy, we have developed the first TRP-based nanotubes, using the LRV and HEAT TRP families; we designed a single SSE from each family to self-assemble in a superhelical fashion. We used structural parameters extrapolated from the parent crystal structures to evaluate the efficacy of our design strategy. Cryo-EM was used to generate atomic models of the peptide nanotubes. In the case of LRV, the helical pitch, handedness, and number of constituent SSEs present in each superhelical turn differed from the crystal structure-based parameters. The HEAT-based design was much more effective, with the helical pitch, handedness, and number of SSEs per superhelical turn closely matching the predicted structure. A dimeric peptide derivative of each of these designs was then evaluated using the same criteria; interestingly, the LRV_dimer assembled in a similar fashion to the parent peptide and the HEAT_dimer did not. A hexameric concatemer of the HEAT peptide sequence was bacterially expressed, purified and assembled. Low- and medium-resolution techniques (CD and TEM, SAXS and STEM, respectively) were used to compare the resultant nanotubes to those generated from the peptide constructs. At medium-resolution, the hexameric and monomeric assemblies were indistinguishable; however, the hexameric nanotube atomic model could not be solved using cryo-EM due to inherent plasticity of the structure. The outside of the HEAT nanotubes was functionalized using a SpyTag:SpyCatcher genetic fusion technique; an octameric HEAT protein was conjugated with SpyTag (ST_HEAT), and the fluorescent protein mCherry was conjugated with SpyCatcher (mCherry_SC). Post-assembly, the ST_HEAT nanotubes were introduced to the mCherry_SC construct, resulting in the functionalization of the convex surface of the HEAT nanotubes. Preliminary structural data obtained from fluorescence microscopy and CLEM indicated the successful functionalization of the nanotube. Computational design has long been paired with rational design strategies to generate helical filaments. Three generations of computational design were applied to a helical hairpin motif, resulting in a more refined approach. Each of these design strategies was evaluated based on the solubility of the sequences, propensity to form helical filaments, and fidelity to the computational model. The most effective strategy for generating helical filaments was to redesign a known assembly, rather than building one from scratch.
Table of Contents
Chapter I: Introduction…………………………………………………………………………….1
1.1 The Significance of Self-Assembly…………………………………………………...1
1.2 Native Helical Assemblies…………………………………………………………….2
1.3 Non-Native Helical Assemblies of Synthesized Peptides…………………………….9
1.4 Helical Assemblies Beyond Biomacromolecules……………………………………18
1.5 Conclusion…………………………………………………………………………...23
1.6 References……………………………………………………………………………25
Chapter II: Ambidextrous Helical Nanotubes from Self-Assembly of Designed Helical Hairpin Motifs………...30
2.1 Self-Assembly of Phage-Mimetic Peptides……………………………………….....30
2.2 Materials and Methods……………………………………………………………….33
2.3 Design, Synthesis, and Biophysical Characterization of the LRV and HEAT Peptide Nanotubes……..38
2.4 Conclusion…………………………………………………………………………...73
2.5 References……………………………………………………………………………76
Chapter III: Concatenation and Functionalization of Tandem-Repeat Protein-Based Helical Nanotubes……..85
3.1 Increasing Assembly Complexity Necessitates Concatenation……………………...85
3.2 HEAT_6R: A TRP Concatemer……………………………………………………..86
3.3 mCherry_HEAT: Design of a Functional Nanotube………………………………...95
3.4 Conclusion………………………………………………………………………….108
3.5 Materials and Methods……………………………………………………………...108
3.6 References…………………………………………………………………………..125
Chapter IV: Computational Design of Helical Nanotubes……………………………………..129
4.1 Computational Versus Rational Versus De Novo Design………………………….129
4.2 TERM-based Designs and the Sol Series…………………………………………..132
4.3 Helical Assembly Builder…………………………………………………………..134
4.4 PDCC and GGHEAT……………………………………………………………….141
4.5 Conclusion………………………………………………………………………….147
4.6 Materials and Methods……………………………………………………………...149
4.6 References…………………………………………………………………………..153
Chapter V: Conclusion………………………………………………………………………….157
Appendix- Re-use Acknowledgements…………………………………………………………162
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