SUMMARY Botulinum neurotoxins (BoNTs, serotypes A-G), elaborated by Clostridium botulinum, can induce lethal paralysis and are classified as category-A bioterrorism agents. However, how BoNTs translocate from endosomes into the cytosol of neurons to gain access to their intracellular targets remains enigmatic. We discovered that binding to the ganglioside GT1b, a toxin co-receptor, enables BoNT/B to sense low pH, undergo a significant change in secondary structure, and transform into a hydrophobic oligomeric membrane protein. Imaging of the toxin on lipid bilayers using atomic force microscopy revealed donut-shaped channel-like structures that resemble other protein translocation assemblies. Toosendanin, a drug with therapeutic effects against botulism, inhibited GT1b-dependent BoNT/B oligomerization, and in parallel truncated BoNT/B single-channel conductance, suggesting that oligomerization plays a role in the translocation reaction. Thus, BoNT/B functions as a coincidence detector for receptor and low pH to ensure spatial and temporal accuracy for toxin conversion into a translocation channel.

Saltatory conduction of nerve impulses along axonal membranes depends on the presence of a multilayered membrane, myelin, that wraps around the axon. Myelin basic protein (MBP) and myelin protein 2 (P2) are intimately involved in the generation of the myelin sheath. They are also implicated in a number of neurological diseases, including autoimmune diseases of both the central and peripheral nervous systems. Here, we have used atomic force microsopy (AFM) to study the effects of MBP and P2 on lipid bilayers. MBP in association with a mica substrate appeared unstructured, and tended to coat the mica surface in the form of a monolayer. In contrast, P2 appeared as discrete particles, with molecular volumes consistent with the formation of both monomers and dimers. Either MBP or P2, at micromolar concentrations, caused stacking of brain lipid bilayers. This stacking effect was significantly potentiated when both proteins were added together. Bilayers composed of phosphatidylcholine (PC) and phosphatidylserine (PS) were stacked by MBP, provided that cholesterol was also present; in contrast, P2 did not stack PC/PS/cholesterol bilayers. Hence, the bilayer stacking effects of the two proteins have different lipid requirements.

Endophilin A is a key player in clathrin-mediated endocytosis at nerve terminals and is essential for the maintenance of synaptic transmission. Endophilin consists of two regions: an SH3 domain that interacts with other endocytotic proteins and an N-BAR domain that binds and bends membranes. Here, we used atomic force microscopy (AFM) under fluid to examine the interaction of the endophilin N-BAR domain with planar supported lipid bilayers, under conditions that closely mimic the environment in which this protein normally operates. We found that when bound to lipid bilayers, the N-BAR domain formed aggregates of various sizes. The N-BAR domain also perturbed the structure of the planar bilayer, at a low concentration (0.15 microM) causing bilayer thinning, and at a 10-fold higher concentration (1.5 microM) forming thin slivers from the bilayer sheet. This bilayer sculpting effect crucially involved the central appendage domain. Reduced hydrophobicity in this domain, caused by the A66D mutation, almost abolished the ability of the endophilin N-BAR domain to bind to supported bilayers. In contrast, increased hydrophobicity, caused by the A66W mutation, switched the bilayer sculpting effect of the N-BAR domain from sliver formation to vesiculation. By following the action of the endophilin N-BAR domain under near-physiological conditions, we have been able to provide additional insights into its membrane binding and bending mechanism.

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HighlightsSso7d-Taq fusion protein purified using a single step of aqueous Two-Phase Extraction (ATPE) is >95% pure and is active.The S-Taq protein has higher thermostability and detergent tolerance over regular Taq polymerase and can be used for PCR's from direct whole blood.The PCR efficiency rate of S-Taq is higher than Taq polymerase and can be used to detect DNA viruses in a clinical setting efficiently.S-Taq can tolerate higher concentrations of magnesium ions and can be used for in-situ PCR’s.S-Taq can be used to carry out PCR’s of bacterial recombinants directly from the overnight culture since it is resistant to inhibition to Luria Bertani broth. This unique property of S-Taq will enable researchers to screen recombinants without the need to isolate the plasmid DNA of recombinants. This would be a huge cost savings for companies engaged in molecular biology research involving PCR’s.