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Mechanisms of translocation-coupled protein unfolding using anthrax toxin as a model


Molecular machines face a number of challenges in transporting a protein either across a membrane or into a proteolytic complex. In many cases, a substrate protein must first be unfolded before being transported through a narrow channel. Despite its importance and relevance to a variety of different processes in the cell, translocation-coupled protein unfolding is still not well understood. In effort to determine the general biophysical mechanisms of this process, anthrax toxin is used as a model system.

In order to understand how a protein unfolds on a translocase channel, planar lipid bilayer electrophysiology, site-directed mutagenesis and thermodynamic stability studies were used to first identify the barriers in the translocation pathway and determine which barrier corresponds to substrate unfolding. Working under conditions where substrate unfolding is rate-limiting, we were then able to map how LFN actually unfolds on the surface of the PA channel.

Next, the role of the channel in substrate unfolding and translocation is discussed. In particular, a novel substrate binding site on the surface of PA was identified from the crystal structure of a PA octamer bound to four LFN substrates. This structure, which was solved by my colleague, Geoffrey Feld, reveals that the first α helix and β strand of each LFN unfold and dock into a deep amphipathic cleft, termed the α clamp. Through extensive mutatgenesis studies on both PA and LFN, Geoff and I determined that this site can bind a broad array of polypeptide substrates. The role of the α clamp in substrate unfolding, channel oligomerization and translocation is investigated and discussed.

Finally, in effort to further probe the α clamp's role in translocation, binding to the site is disrupted and the effects on translocation are investigated. Preliminary hypotheses and future directions are discussed.

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