RESUMO
Clostridium botulinum type A neurotoxin (BoNT/A complex) is of great interest to the pharmaceutical industry. The drug itself is a natural complex of the toxin and a number of associated proteins. Surprisingly, relatively little is known about the exact structure and stability of the 900 kDa BoNT/A complex and its component proteins with the exception of the 150 kDa neurotoxin. In this study we describe the relative stability of the BoNT/A complex, the neurotoxin, and its associated proteins over a wide range of temperature and pH employing circular dichroism, intrinsic and 8-anilino-1-naphthalene sulfonate (ANS) fluorescence, and static light scattering. The data suggest a strong stabilizing effect of the associated proteins on the neurotoxin component. This data is compiled into empirical phase diagrams which permit the simultaneous visualization of multiple data sets over a wide range of conditions.
Assuntos
Clostridium botulinum tipo A/química , Clostridium botulinum tipo A/metabolismo , Neurotoxinas/química , Neurotoxinas/metabolismo , Naftalenossulfonato de Anilina/metabolismo , Dicroísmo Circular , Clostridium botulinum tipo A/isolamento & purificação , Estabilidade de Medicamentos , Corantes Fluorescentes/metabolismo , Concentração de Íons de Hidrogênio , Luz , Peso Molecular , Neurotoxinas/isolamento & purificação , Espalhamento de Radiação , Espectrometria de Fluorescência , TemperaturaRESUMO
The high affinity of certain cellular polyanions for many proteins (polyanion-binding proteins (PABPs)) has been demonstrated previously. It has been hypothesized that such polyanions may be involved in protein structure stabilization, stimulation of folding through chaperone-like activity, and intra- and extracellular protein transport as well as intracellular organization. The purpose of the proteomics studies reported here was to seek evidence for the idea that the nonspecific but high affinity interactions of PABPs with polyanions have a functional role in intracellular processes. Utilizing yeast protein arrays and five biotinylated cellular polyanion probes (actin, tubulin, heparin, heparan sulfate, and DNA), we identified proteins that interact with these probes and analyzed their structural and amino acid sequence requirements as well as their predicted functions in the yeast proteome. We also provide evidence for the existence of a network-like system for PABPs and their potential roles as critical hubs in intracellular behavior. This investigation takes a first step toward achieving a better understanding of the nature of polyanion-protein interactions within cells and introduces an alternative way of thinking about intracellular organization.