UC Berkeley

It is imperative to investigate protein energy landscapes within the framework of physiological or near-physiological conditions, particularly when emphasizing the significance of the protein folding/unfolding process or of specific unfolded states in relation to a protein's biological function.Ubiquitination, a post-translational modification instrumental in proteasomal degradation of protein substrates, can influence protein stability. Proteins experience force-mediated denaturation when unfolded by the proteasome; therefore, I employed single-molecule force spectroscopy to study rates of unfolding of ubiquitinated proteins. I demonstrate that ubiquitination of the model protein barstar at lysine 60 not only increases its force-induced unfolding rate, but also influences the responsiveness of unfolding rate to force, implicating a change in unfolding pathways. Human γD-crystallin is a monomeric protein abundant in the eye lens nucleus that must remain stably folded for an individual’s entire lifetime to avoid aggregation and cataract formation. It is not clear whether intermediates of γD-crystallin populated under denaturing conditions are also populated under native conditions, or what relevance they hold to the mechanism of aggregation. Therefore, I employed hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) to characterize several variants of γD-crystallin under both native and denaturing conditions. By investigating several types of mutations in each of γD-crystallin’s two domains, I show that, for two cataract- prone variants of γD-crystallin, the lowest-energy equilibrium intermediate populated under native conditions is structurally and energetically distinct to the intermediate populated under chemically denaturing conditions. The interface between the two domains is crucial to the formation of this new intermediate, and disruption of the interface either by mutation or by mild denaturation permits direct observation of both intermediates at the same time. The natively populated partially folded conformation of γD-crystallin exposes a surface which is normally buried both in the full-length structure and in isolated folded domains. Together with the noted effects of mutations, this suggests it may be significant to aggregation and to cataract formation.