ClpX as a Tool for Assessing the Impact of Rate and Direction of Vectorial Protein Appearance on Folding Outcomes

In the cell, protein folding can start from N- to C-terminus, as a protein is synthesized by the ribosome. Vectorial appearance enables proteins to fold progressively, exploring an energy landscape that increases in size and complexity as the polypeptide chain length increases. However, little is known about the impact of vectorial appearance on protein folding mechanisms, including the extent to which vectorial appearance may favor the formation of folding intermediates that are more (or less) likely to fold to the native, functional structure, versus misfolding and aggregation. Most of our current understanding of protein folding mechanisms is derived from in vitro experiments initiated by diluting a full-length protein out of a chemical denaturant, a scenario that enables folding to begin with interactions formed between any two portions of the entire polypeptide chain. Currently, no method exists to directly compare the impact of initiating folding from the N- versus C-terminus. Here, I adapted the ring-shaped AAA+ translocase ClpX to induce vectorial protein appearance from either terminus via translocation through the central pore of ClpX and used this approach to study the impact of vectorial appearance on folding outcomes for two fluorescent proteins. In Chapter 3, I implement a competitive inhibition strategy to show that native proteins that are unfolded and translocated (from C- to N- terminus) by ClpX can refold after extrusion out of the central pore of the translocase. In Chapter 4, I tested covalent linkage strategies for the attachment of an N-terminal ClpX affinity tag, ultimately identifying a SpyCatcher-based approach as the most efficient strategy. Finally, in Chapter 5, I studied the effect that vectorial appearance in opposite directions has on folding outcomes of pre-unfolded proteins. I conclude that vectorial appearance significantly affects folding in a direction-specific manner, and the rate of chain appearance further influences folding outcomes. Moreover, although the fluorescent proteins have very similar native structures, their folding mechanisms were distinctly sensitive to vectorial appearance. In summary, this body of work highlights the ruggedness of protein folding energy landscapes, including unique features for structurally homologous proteins, and demonstrates directly that the success of protein folding is sensitive to how the protein is introduced to its folding energy landscape.