All-atom molecular dynamics simulations combined with graph-theoretic analysis reveal that clustering of monomethyl phosphate dianion (MMP) is strongly influenced by the types and combinations of cations in the aqueous solution. Although Ca promotes the formation of stable and large MMP clusters, K alone does not. Nonetheless, clusters are larger and their link lifetimes are longer in mixtures of K and Ca. This "synergistic" effect depends sensitively on the Lennard-Jones interaction parameters between Ca and the phosphorus oxygen and correlates with the hydration of the clusters. The pronounced MMP clustering effect of Ca in the presence of K is confirmed by Fourier transform infrared spectroscopy. The characterization of the cation-dependent clustering of MMP provides a starting point for understanding cation-dependent clustering of phosphoinositides in cell membranes.

Clostridium botulinum strains secrete their neurotoxins (BoNT) along with a group of neurotoxin-associated proteins (NAPs) that enhance the oral toxicity and provide protection to the neurotoxin against acidity, temperature and proteases in the G.I. tract. A major component of NAPs is Hn-33, a 33kDa protein, which is also protease resistant and strongly protects BoNT. The complex form of BoNT/A is used as a commercial therapeutic formulation against many neuromuscular disorders and for cosmetic purposes. Immune response against this formulation could hinder its long-term use; therefore, it is important to characterize the immunological properties of the associated proteins. This study aims to understand the immunological reactivity of BoNT/A complex, BoNT, NAPs, and Hn-33 through a series of competitive enzyme-linked immunosorbent assays (ELISA). The results indicated that BoNT/A complex competed 6 times more with complex antibodies compared to the neurotoxin confirming that the higher immunogenicity of BoNT/A complex was indeed a result of the associated proteins with the neurotoxin complex. While the nearly identical immuno-reactivity of BoNT/A complex and Hn-33 with Hn-33 antibodies indicated that the reactivity was due to the higher immunogenicity not the abundance of Hn-33 in the complex. Both the ELISA and immuno-blot results implied that Hn-33 is primarily responsible for eliciting the antibody response in BoNT/A complex, therefore it may be possible to employ Hn-33 as an adjuvant for development of vaccines against botulism.

Seven distinct strains of Clostridium botulinum (type A to G) each produce a stable complex of botulinum neurotoxin (BoNT) along with neurotoxin-associated proteins (NAPs). Type A botulinum neurotoxin (BoNT/A) is produced with a group of NAPs and is commercially available for the treatment of numerous neuromuscular disorders and cosmetic purposes. Previous studies have indicated that BoNT/A complex composition is specific to the strain, the method of growth and the method of purification; consequently, any variation in composition of NAPs could have significant implications to the effectiveness of BoNT based therapeutics. In this study, a standard analytical technique using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and densitometry analysis was developed to accurately analyze BoNT/A complex from C. botulinum type A Hall strain. Using 3 batches of BoNT/A complex the molar ratio was determined as neurotoxin binding protein (NBP, 124 kDa), heavy chain (HC, 90 kDa), light chain (LC, 53 kDa), NAP-53 (50 kDa), NAP-33 (36 kDa), NAP-22 (24 kDa), NAP-17 (17 kDa) 1:1:1:2:3:2:2. With Bradford, Lowry, bicinchoninic acid (BCA) and spectroscopic protein estimation methods, the extinction coefficient of BoNT/A complex was determined as 1.54 ± 0.26 (mg/mL)(-1)cm(-1). These findings of a reproducible BoNT/A complex composition will aid in understanding the molecular structure and function of BoNT/A and NAPs.

No abstract

into the nanoparticles the interfacial adsorption capability of the complex increased slightly depending on the palmitoylation state of the protein. We have also analyzed the interaction of the complexes with surfactant monolayers and bilayers. Fluorescently labeled nanocomplexes seem to associate to both lipid structures with no apparent lipid transfer. These nanostructures could then constitute an efficient vehicle to direct molecules such as proteins or drugs to the respiratory air-liquid interface.