Protein dynamics are critical to the structure and function of proteins. However, due to the complexity they inherently bring to the protein design problem, dynamics historically have not been considered in computational protein design (CPD). Herein, we present meta-MSD, a new CPD methodology for the design of protein dynamics. We applied our methodology to the design of a novel mode of conformational exchange in Streptococcal protein G domain B1, producing dynamic variants we termed DANCERs. Predictions were validated by NMR characterization of selected DANCERs, confirming that our meta-MSD framework is suitable for the computational design of protein dynamics. We then performed a thorough NMR characterization of the sequence determinants of dynamics in one DANCER, isolating two mutations responsible for the novel dynamics this protein exhibits. The first, A34F, is responsible for destabilizing the highly stable native Gβ1 conformation, allowing the protein to sample other conformational states. The second, V39L mediates subtle interactions that stabilize the designed conformational trajectory in the context of the A34F mutation. Together, these results highlight the role of protein plasticity in the development of dynamics and the need for highly accurate computational tools to approach similar design problems. Finally, we present an NMR-based characterization of structural dynamics in a family of related red fluorescent proteins (RFPs) and pinpoint regions of the RFP structure where dynamics correlate to RFP brightness. This overview of the RFP dynamics-function relationship will be used in future projects to perform a computation design of functional dynamics in RFPs.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/39211 |
| Date | 16 May 2019 |
| Creators | Damry, Adam |
| Contributors | Chica, Roberto |
| Publisher | Université d'Ottawa / University of Ottawa |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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