Evaluation of a novel cloud-based software platform for structured experiment design and linked data analytics.

Open data in science requires precise definition of experimental procedures used in data generation, but traditional practices for sharing protocols and data cannot provide the required data contextualization. Here, we explore implementation, in an academic research setting, of a novel cloud-based software system designed to address this challenge. The software supports systematic definition of experimental procedures as visual processes, acquisition and analysis of primary data, and linking of data and procedures in machine-computable form. The software was tested on a set of quantitative microbial-physiology experiments. Though time-intensive, definition of experimental procedures in the software enabled much more precise, unambiguous definitions of experiments than conventional protocols. Once defined, processes were easily reusable and composable into more complex experimental flows. Automatic coupling of process definitions to experimental data enables immediate identification of correlations between procedural details, intended and unintended experimental perturbations, and experimental outcomes. Software-based experiment descriptions could ultimately replace terse and ambiguous 'Materials and Methods' sections in scientific journals, thus promoting reproducibility and reusability of published studies.

Digital publishing isn't enough: the case for 'blueprints' in scientific communication.

Since the time of Newton and Galileo, the tools for capturing and communicating science have remained conceptually unchanged - in essence, they consist of observations on paper (or electronic variants), followed by a 'letter' to the community to report your findings. These age-old tools are inadequate for the complexity of today's scientific challenges. If modern software engineering worked like science, programmers would not share open source code; they would take notes on their work and then publish long-form articles about their software. Months or years later, their colleagues would attempt to reproduce the software based on the article. It sounds a bit silly, and yet even, this level of prose-based methodological discourse has deteriorated in science communication. Materials and Methods sections of papers are often a vaguely written afterthought, leaving researchers baffled when they try to repeat a published finding. It's time for a fundamental shift in scientific communication and sharing, a shift akin to the advent of computer-aided design and source code versioning. Science needs reusable 'blueprints' for experiments replete with the experiment designs, material flows, reaction parameters, data, and analytical procedures. Such an approach could establish the foundations for truly open source science where these scientific blueprints form the digital 'source code' for a supply chain of high-quality innovations and discoveries.

Cys-Cys cross-linking shows contact between the N-terminus of lethal factor and Phe427 of the anthrax toxin pore.

Electrophysiological studies of wild-type and mutated forms of anthrax protective antigen (PA) suggest that the Phe clamp, a structure formed by the Phe427 residues within the lumen of the oligomeric PA pore, binds the unstructured N-terminus of the lethal factor and the edema factor during initiation of translocation. We now show by electrophysiological measurements and gel shift assays that a single Cys introduced into the Phe clamp can form a disulfide bond with a Cys placed at the N-terminus of the isolated N-terminal domain of LF. These results demonstrate direct contact of these Cys residues, supporting a model in which the interaction of the unstructured N-terminus of the translocated moieties with the Phe clamp initiates N- to C-terminal threading of these moieties through the pore.

Trapping a translocating protein within the anthrax toxin channel: implications for the secondary structure of permeating proteins.

Anthrax toxin consists of three proteins: lethal factor (LF), edema factor (EF), and protective antigen (PA). This last forms a heptameric channel, (PA(63))(7), in the host cell's endosomal membrane, allowing the former two (which are enzymes) to be translocated into the cytosol. (PA(63))(7) incorporated into planar bilayer membranes forms a channel that translocates LF and EF, with the N terminus leading the way. The channel is mushroom-shaped with a cap containing the binding sites for EF and LF, and an ∼100 Å-long, 15 Å-wide stem. For proteins to pass through the stem they clearly must unfold, but is secondary structure preserved? To answer this question, we developed a method of trapping the polypeptide chain of a translocating protein within the channel and determined the minimum number of residues that could traverse it. We attached a biotin to the N terminus of LF(N) (the 263-residue N-terminal portion of LF) and a molecular stopper elsewhere. If the distance from the N terminus to the stopper was long enough to traverse the channel, streptavidin added to the trans side bound the N-terminal biotin, trapping the protein within the channel; if this distance was not long enough, streptavidin did not bind the N-terminal biotin and the protein was not trapped. The trapping rate was dependent on the driving force (voltage), the length of time it was applied, and the number of residues between the N terminus and the stopper. By varying the position of the stopper, we determined the minimum number of residues required to span the channel. We conclude that LF(N) adopts an extended-chain configuration as it translocates; i.e., the channel unfolds the secondary structure of the protein. We also show that the channel not only can translocate LF(N) in the normal direction but also can, at least partially, translocate LF(N) in the opposite direction.

Interactions of anthrax lethal factor with protective antigen defined by site-directed spin labeling.

The protective antigen (PA) moiety of anthrax toxin forms oligomeric pores that translocate the enzymatic moieties of the toxin--lethal factor (LF) and edema factor (EF)--across the endosomal membrane of mammalian cells. Here we describe site-directed spin-labeling studies that identify interactions of LF with the prepore and pore conformations of PA. Our results reveal a direct interaction between the extreme N terminus of LF (residues 2-5) and the Φ-clamp, a structure within the lumen of the pore that catalyzes translocation. Also, consistent with a recent crystallographic model, we find that, upon binding of the translocation substrate to PA, LF helix α1 separates from helices α2 and α3 and binds in the α-clamp of PA. These interactions, together with the binding of the globular part of the N-terminal domain of LF to domain 1' of PA, indicate that LF interacts with the PA pore at three distinct sites. Our findings elucidate the state from which translocation of LF and EF proceeds through the PA pore.