A study of coevolved, cofolding and functionally linked proteins and the characterization of amyloid-like fibrils and oligomers towards the understanding of protein aggregation in disease

My work early in the graduate program involved identifying and characterizing interacting proteins that coevolve to be present or absent together across genomes. This assumption lead to bioinformatics predictions that identified the YheNML protein complex as a functional unit involved in protein translation; its subunits require cotranslation in order to cofold and physically assemble into a complex. Its crystal structure was pursued with the hope that its structure will lead to elucidation of function.;I then worked on bovine pancreatic ribonuclease A (RNase A) as a system for studying amyloid fibril formation and protein aggregation that cause cellular dysfunction. I found that short segments from human amyloid and prion proteins were sufficient in converting globular RNase A into the amyloid, fibrillar form. RNase A was found to contain endogenous fibril-forming segments, although normal RNase A never forms fibrils in myriad denaturing and refolding conditions. RNase A fibrils were induced by increasing the flexibility of these endogenous segments. A cross-(3 spine propagates fibril formation and RNase A can maintain native fold within these fibrils. This supports the hypothesis that only a small segment of a protein misfolds and participates in the architecture of the fibril spine. This work also illustrates that non-amyloid proteins can contain fibril-forming segments that, with ample flexibility, can cause fibril formation.;In order to establish the species of amyloid which is toxic to the cell, I investigated the influence of oligomeric and fibrillar forms of Amyloid-13 and AlphaB Crystallin segments on cell vitality. In both cases, the oligomers were more toxic to human neuroblastoma cells than fibrils. These observations support growing evidence that the formation of the fibrils is perhaps a method of sequestering toxic, oligomeric species in the cell.;This work has enabled my collaborators and me to understand the mechanisms of amyloid formation by globular proteins as well as the structural characteristics responsible for cellular toxicity.