Plasmon rulers as dynamic molecular rulers in enzymology.

This chapter provides an introduction to the concept of "plasmon rulers," pairs of biopolymer-linked tethered nanoparticles which act as nonblinking, nonbleaching rulers for dynamic molecular distance measurements. Plasmon rulers utilize the distance dependence of the plasmon coupling between individual noble metal particles to measure distances. Although the plasmon ruler approach is still an emerging technology, proof-of-principle experiments have demonstrated that plasmon rulers can already be used to investigate structural fluctuations in nucleoprotein complexes, monitor nuclease catalyzed DNA or RNA cleavage reactions, and detect DNA bending. The physical concepts underlying plasmon rulers are summarized, and effective assembly approaches as well as recent applications are discussed. Plasmon rulers are a useful addition to the single molecule biophysics toolbox, since they allow single biomolecules to be continuously monitored for days at high temporal resolutions.

Lethal factor unfolding is the most force-dependent step of anthrax toxin translocation.

Cellular compartmentalization requires machinery capable of translocating polypeptides across membranes. In many cases, transported proteins must first be unfolded by means of the proton motive force and/or ATP hydrolysis. Anthrax toxin, which is composed of a channel-forming protein and two substrate proteins, is an attractive model system to study translocation-coupled unfolding, because the applied driving force can be externally controlled and translocation can be monitored directly by using electrophysiology. By controlling the driving force and introducing destabilizing point mutations in the substrate, we identified the barriers in the transport pathway, determined which barrier corresponds to protein unfolding, and mapped how the substrate protein unfolds during translocation. In contrast to previous studies, we find that the protein's structure next to the signal tag is not rate-limiting to unfolding. Instead, a more extensive part of the structure, the amino-terminal beta-sheet subdomain, must disassemble to cross the unfolding barrier. We also find that unfolding is catalyzed by the channel's phenylalanine-clamp active site. We propose a broad molecular mechanism for translocation-coupled unfolding, which is applicable to both soluble and membrane-embedded unfolding machines.