Clostridial neurotoxins, including tetanus toxin and the seven serotypes of botulinum toxin (A-G), are produced as single chains and cleaved to generate toxins with two chains joined by a single disulphide bond (Fig. 1). The heavy chain (M(r) 100,000 (100K)) is responsible for specific binding to neuronal cells and cell penetration of the light chain (50K), which blocks neurotransmitter release. Several lines of evidence have recently suggested that clostridial neurotoxins could be zinc endopeptidases. Here we show that tetanus and botulinum toxins serotype B are zinc endopeptidases, the activation of which requires reduction of the interchain disulphide bond. The protease activity is localized on the light chain and is specific for synaptobrevin, an integral membrane protein of small synaptic vesicles. The rat synaptobrevin-2 isoform is cleaved by both neurotoxins at the same single site, the peptide bond Gln 76-Phe 77, but the isoform synaptobrevin-1, which has a valine at the corresponding position, is not cleaved. The blocking of neurotransmitter release of Aplysia neurons injected with tetanus toxin or botulinum toxins serotype B is substantially delayed by peptides containing the synaptobrevin-2 cleavage site. These results indicate that tetanus and botulinum B neurotoxins block neurotransmitter release by cleaving synaptobrevin-2, a protein that, on the basis of our results, seems to play a key part in neurotransmitter release.

No abstract

Changes in protein conformation can affect protein function, but methods to probe these structural changes on a global scale in cells have been lacking. To enable large-scale analyses of protein conformational changes directly in their biological matrices, we present a method that couples limited proteolysis with a targeted proteomics workflow. Using our method, we assessed the structural features of more than 1,000 yeast proteins simultaneously and detected altered conformations for ~300 proteins upon a change of nutrients. We find that some branches of carbon metabolism are transcriptionally regulated whereas others are regulated by enzyme conformational changes. We detect structural changes in aggregation-prone proteins and show the functional relevance of one of these proteins to the metabolic switch. This approach enables probing of both subtle and pronounced structural changes of proteins on a large scale.

The folding of a polypeptide chain of a relatively large globular protein into its unique three-dimensional and functionally active structure occurs via folding intermediates. These partly folded states of proteins are difficult to characterize, because they are usually short lived or exist as a distribution of possible conformers. A variety of experimental techniques and approaches have been utilized in recent years in numerous laboratories for characterizing folding intermediates that occur at equilibrium, including spectroscopic techniques, solution X-ray scattering, calorimetry and gel filtration chromatography, as well as genetic methods and theoretical calculations. In this review, we focus on the use of proteolytic enzymes as probes of the structure and dynamics of folding intermediates and we show that this simple biochemical technique can provide useful information, complementing that obtained by other commonly used techniques and approaches. The key result of the proteolysis experiments is that partly folded states (molten globules) of proteins can be sufficiently rigid to prevent extensive proteolysis and appear to maintain significant native-like structure.

Limited proteolysis experiments can be successfully used to probe conformational features of proteins. In a number of studies it has been demonstrated that the sites of limited proteolysis along the polypeptide chain of a protein are characterized by enhanced backbone flexibility, implying that proteolytic probes can pinpoint the sites of local unfolding in a protein chain. Limited proteolysis was used to analyze the partly folded (molten globule) states of several proteins, such as apomyoglobin, alpha-lactalbumin, calcium-binding lysozymes, cytochrome c and human growth hormone. These proteins were induced to acquire the molten globule state under specific solvent conditions, such as low pH. In general, the protein conformational features deduced from limited proteolysis experiments nicely correlate with those deriving from other biophysical and spectroscopic techniques. Limited proteolysis is also most useful for isolating protein fragments that can fold autonomously and thus behave as protein domains. Moreover, the technique can be used to identify and prepare protein fragments that are able to associate into a native-like and often functional protein complex. Overall, our results underscore the utility of the limited proteolysis approach for unravelling molecular features of proteins and appear to prompt its systematic use as a simple first step in the elucidation of structure-dynamics-function relationships of a novel and rare protein, especially if available in minute amounts.