Towards the Structure of Yeast Prions
- Author(s): Chu, Clement SM;
- Advisor(s): Weissman, Jonathan S;
- et al.
The yeast prion [PSI+] is caused by the self-propagation of beta-sheet rich aggregates of the translation termination factor Sup35. Studies in this system have shown some of the most definitive proof of the protein-only nature of prions and have explained the strain phenomenon by showing that heritable information can be encoded by the conformation of a protein. The structural basis of how these various protein conformations lead to phenotypic differences has been difficult to discern primarily due to the technical difficulties of working with an aggregating protein. The use of non-perturbative stable isotopic labels and solid-state NMR (SSNMR) has been successful in characterizing some semi-ordered protein aggregates. However, the Gln/Asn-rich nature and low sequence complexity of Sup35's prion domain has prevented sequence specific information from being generated by SSNMR rendering this technique less useful. The work presented here was primarily aimed at generating a tool to allow for site-specific isotopic labeling of SupNM. I describe a general strategy for the semisynthesis of Gln/Asn-rich proteins utilizing optimized chemical synthesis, recombinant protein production, and native chemical ligation. I show that semisynthetic SupNM is capable of forming amyloid fibers that are competent to infect yeast. Utilizing this method will allow for precise placement of isotopic labels and enable residue-specific chemical shift assignments in SSNMR with the goal of generating high-resolution molecular models of the [PSI+] prion.