We have made an immobilized and reusable molecular chaperone system for oxidative refolding chromatography. Its three components-GroEL minichaperone (191-345), which can prevent protein aggregation; DsbA, which catalyzes the shuffling and oxidative formation of disulfide bonds; and peptidyl-prolyl isomerase-were immobilized on an agarose gel. The gel was applied to the refolding of denatured and reduced scorpion toxin Cn5. The 66-residue toxin, which has four disulfide bridges and a cis peptidyl-proline bond, had not previously been refolded in reasonable yield. We recovered an 87% yield of protein with 100% biological activity.

Potassium-channel-blocking scorpion toxins (A-K-toxins) have been shown to be valuable tools for the study of potassium channels. Here we report two toxins, cobatoxin 1 and 2, of 32 amino acids, containing three disulphide bridges, that were isolated from the venom of the Mexican scorpion Centruroides noxius. Their primary sequences show less than 40% identity to other A-K-toxins. It is therefore proposed that they belong to subfamily 9. The cDNA of cobatoxin 1 encodes a putative signal peptide, a putative short propeptide, the mature peptide and two amino acids that are processed to leave cobatoxin 1 amidated at the C-terminus. In rat brain synaptosomal membranes cobatoxin 1 and cobatoxin 2 bind to a common binding site of A-K-toxins with K i values of 109 pM and 87 pM, respectively. Moreover, they block the Shaker and K V 1.1 K ϩ channels with moderate affinities, with K d values of around 0.7 µM and 4.1 µM (Shaker) and 0.5 µM and 1.0 µM (K V1.1), respectively. A three-dimensional model of cobatoxin 1 was generated and used to interpret the obtained functional data on a structural basis.Keywords : A-K-toxin ; Centruroides noxius ; cobatoxin; potassium channel; scorpion toxin.Potassium channels form a large family of integral mem-surements and mutational analyses, that they block K ϩ channels from the external side, occluding the channel pore [19Ϫ22]. Disbrane proteins that are present in almost every living cell. They play a key physiological role by setting the resting potential and placement experiments on rat brain synaptosomes provide evidence that the receptor site is shared by mast-cell-degranulating shaping bioelectric signals [1,2] PVIIA [27]. Thus, despite these peptides presenting very different three-dimensional folds [14, 28Ϫ34], they seem to act on Neurotoxins that selectively and sensitively block potassium channels (K-toxins) form a structurally diverse group, from K ϩ channels in a similar way. The only exception described has been hanatoxin [35,36]. small peptides to rather complex proteins. Some of them have been shown to be valuable pharmacological tools to study the Scorpion K-toxins were successfully used to identify the pore-forming region of K ϩ channels [20,37], to clarify the strucmolecular structure, function and diversity of K ϩ channels [8,9]. They can be grouped, based on the similarity of their size tural subunit stoichiometry [38], and to identify the functional stoichiometry of inactivation of K ϩ channels [39]. They served and disulphide-bond pattern, into seven groups, which generally reflect their natural origin : scorpion toxins of 31Ϫ39 amino as molecular calipers to measure the dimension of the outer vestibule of the pore of particular channels [40Ϫ47]. Furthermore, acids [9]; dendrotoxins from mamba snakes and kalicludines from sea anemone, of 57Ϫ60 amino acids [10Ϫ12] ; small pep-they were used for the purification, isolation [24, 48Ϫ50] and pharmacological classification of K ϩ channels [8], and to study tides of 18Ϫ22 amino acids isolated from bee venom, including ma...

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Global environmental problems such as climate change are not bounded by national borders or scientific disciplines, and therefore require international, interdisciplinary teamwork to develop understandings of their causes and solutions. Interdisciplinary scientific work is difficult enough, but these challenges are often magnified when teams also work across national boundaries. The literature on the challenges of interdisciplinary research is extensive. However, research on international, interdisciplinary teams is nearly non-existent. Our objective is to fill this gap by reporting on results from a study of a large interdisciplinary, international National Science Foundation Partnerships for International Research and Education (NSF-PIRE) research project across the Americas. We administered a structured questionnaire to team members about challenges they faced while working together across disciplines and outside of their home countries in Argentina, Brazil, and Mexico. Analysis of the responses indicated five major types of barriers to conducting interdisciplinary, international research: integration, language, fieldwork logistics, personnel and relationships, and time commitment. We discuss the causes and recommended solutions to the most common barriers. Our findings can help other interdisciplinary, international research teams anticipate challenges, and develop effective solutions to minimize the negative impacts of these barriers to their research.

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