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 β-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

Hermeneutics, accreting receptions, hypermedia: A tool for reference versus a tool for instruction

We provide a select overview of tools supporting traditional Jewish learning. Then we go on to discuss our own HyperJoseph/HyperIsaac project in instructional hypermedia. Its application is to teaching, teacher training, and self-instruction in given Bible passages. The treatment of two narratives has been developed thus far. The tool enables an analysis of the text in several respects: linguistic, narratological, etc. Moreover, the Scriptures' focality throughout the cultural history makes this domain of application particularly challenging, in that there is a requirement for the tool to encompass the accretion of receptions in the cultural repertoire, i.e., several layers of textual traditions—either hermeneutic (i.e., interpretive), or appropriations—related to the given core passage, thus including "secondary" texts (i.e., such that are responding or derivative) from as disparate realms as Roman-age and later homiletics, Medieval and later commentaries or supercommentaries, literary appropriations, references to the arts and modern scholarship, etc. in particular, the Midrash (homiletic expansions) is adept at narrative gap filling, so the narratives mushroom at the interstices where the primary text is silent. The genealogy of the project is rooted in Weiss' index of novelist Agnon's writings, which was eventually upgraded into a hypertextual tool, including Agnon's full-text and ancillary materials. Those early tools being intended primarily for reference and research-support in literary studies, the Agnon hypertext system was initially emulated in the conception of HyperJoseph, which is applied to the Joseph story from Genesis. Then, the transition from a tool for reference to an instructional tool required a thorough reconception in an educational perspective, which led to HyperIsaac, on the sacrifice of Isaac, and to a redesign and upgrade of HyperJoseph as patterned after HyperIsaac