Goldenberg 9316065 The goal of this project is to determine what factors influence the folding and disulfide-bond formation of a small peptide toxin found in the venom of a marine snail, Conus magus. The venoms are composed of small peptides, typically 12 to 30 residues long, most of which contain two or three disulfide bonds. Although these molecules are much smaller than typical globular proteins, they fold into well-defined three-dimensional structures. The small size of the peptides and the high degree of sequence variability suggests that information in addition to that found in the mature toxin may be necessary to direct correct folding. This information may be provided by recently identified pro-peptide sequences. Folding may also be influenced by the enzyme protein disulfide isomerase (PDI) or other cellular components. In order to determine the role of the pro-peptide sequence, the in vitro folding and disulfide formation of the mature toxin will be compared with that of the pro-toxin form produced in E. coli. Comparing the rates of folding and the distribution of intermediates with and without the pro-peptide will indicate whether the pro-peptide can increase the rates of productive folding. The folding kinetics of the mature and pro-toxin forms will be examined in the presence of PDI. An in vitro translation-translocation system will also be used to study the folding of the toxin precursor. Comparison with results obtained in vivo will indicate how well the in vitro reactions mimic folding in vivo and may reveal the presence of other components of the endoplasmic reticulum that are important for folding. %%% Proteins are long linear molecules made up of amino acids linked together by chemical bonds. In this regard, proteins resemble synthetic polymers such as nylon. Proteins differ from most other polymers, however, because the chains are able to fold into unique three-dimensional structures. The shape of the folded protein and the distribution of chemical groups allow proteins to interact with other molecules with high specificity. Most proteins known to fold into a unique structure contain a minimum of about 50 amino acids. A very interesting exception to this generalization has been discovered in peptides found in the venoms of snails in the genus Conus. This genus is made up of about 500 species of cone-shaped marine snails. These snails are predators and are able to paralyze their prey by injecting highly potent venoms containing mixtures of small peptides. These peptides are typically only 12 to 30 amino acids long, but have been shown to be folded into well-defined three-dimensional structures. The different peptides interact strongly and specifically with components of the nervous system. The high specificities of these interactions have made the Conus toxins powerful tools for studying the molecular components of the nervous system. The goal of this project is to determine what molecular and cellular factors enable the Conus peptides to fold into their specific conformations, in spite of their small size. In addition to helping to elucidate the details of this unusual biological system, the results of these studies may be useful in the design and production of artificial peptides with novel biological activities. ***
