Fluorescence Methods Applied to the Description of Urea-Dependent YME1L Protease Unfolding

Abstract

ATP-dependent proteases are ubiquitous across all kingdoms of life and are critical to the maintenance of intracellular protein quality control. The enzymatic function of these enzymes requires structural stability under conditions that may drive instability and/or loss of function in potential protein substrates. Thus, these molecular machines must demonstrate greater stability than their substrates in order to ensure continued function in essential quality control networks. We report here a role for ATP in the stabilization of the inner membrane YME1L protease. Qualitative fluorescence data derived from protein unfolding experiments with urea reveal non-standard protein unfolding behavior that is dependent on [ATP]. Using multiple fluorophore systems, stopped-flow fluorescence experiments demonstrate a depletion of the native YME1L ensemble by urea-dependent unfolding and formation of a non-native conformation. Additional stopped-flow fluorescence experiments based on nucleotide binding and unfoldase activities predict that unfolding yields significant loss of active YME1L hexamers from the starting ensemble. Taken together, these data clearly define the stress limits of an important mitochondrial protease.

Examination of the Role of Mg2+ in the Mechanism of Nucleotide Binding to the Monomeric YME1L AAA+ Domain

“…Taken together, our data support a model in which oxidative stress may promote ATP binding by an increased level of sampling of conformational arrangements competent for nucleotide binding. This presents a mechanism by which YME1L binding of nucleotide may allow for escape from OMA1 detection or stabilization against intracellular stress exposure, 19 thereby allowing for continued function in protein quality control and continued maintenance of mitochondrial morphology.…”