We report the synthesis and evaluation of (EDTA-2-aminoethyl) 2-pyridyl disulfide. By using this easily prepared cysteine-specific hydrophilic reagent, an ethylenediaminetriacetic acid-Fe3+ complex (EDTA-Fe) was covalently attached to a single genetically engineered cysteine residue in staphylococcal nuclease. Upon addition of the iron reductant ascorbate, the nuclease-EDTA-Fe conjugate underwent a protein self-cleavage reaction mediated by reactive oxygen species. Sequence analysis of the products indicated that cleavage occurs close in tertiary structure to the EDTA-Fe attachment site. In the presence of denaturants, the cleavage pattern changes and the reaction is limited to residues proximal in sequence to the cysteine attachment site. These results indicate that intramolecular protein cleavage reactions mediated by EDTA-Fe can be used to evaluate changes in protein conformation. The reagent described should be a useful tool in the structural mapping of nonnative protein states populated at equilibrium, such as the molten globule, that are frequently refractory to conventional structure analysis.Here, we report the synthesis and evaluation of (EDTA-2-aminoethyl) 2-pyridyl disulfide (EPD; Fig. 1, compound 1) an easily prepared cysteine-specific hydrophilic reagent useful for reversibly conjugating ethylenediaminetriacetic acid to any free thiol group in a macromolecule. This reagent is a versatile tool that promotes intramolecular and localized protein cleavage. Staphylococcal nuclease was genetically engineered to introduce a single cysteine at position 28, and this variant, K28C, was used to characterize EPD-Fe as a protein cleavage reagent. Experimentation with a protein of known three-dimensional structure (20, 21) allowed the accessibility and proximity of cleavage sites to be assessed. Sequence analysis of K28C-EDTA-Fe fragmentation products identified several cleavage sites located close in tertiary structure to the reagent attachment site. In the presence of sodium dodecyl sulfate (SDS) or guanidinium chloride, cleavage at sites remote in linear sequence was not observed and the reaction was limited to residues proximal in sequence to the Cys-28 attachment site.There is an increasing need for new probes to study the topology of protein nonnative states, such as the molten globule and other folding intermediates, that can be populated at equilibrium (1, 2). Modem NMR techniques are revolutionizing the analysis of small proteins in solution, but they can be used to characterize partially folded molecules only under favorable conditions (3). Antibodies, proteases, and chemical probes have been used in the study of protein folding (4-8), but they are not well suited for mapping partially folded structure.A new class of chemical probes was devised largely for footprinting studies of DNA (9, 10). These reagents generate reactive oxygen species that label surrounding structural elements by oxidative degradation. Several metal chelates, bound covalently (11-13) or by affinity (14-16) to a protein, are...

Chemical cleavage with reactive oxygen species generated by EPD-Fe, a protein-tethered EDTA-Fe reagent, has been proposed as a method to map the structure of nonnative equilibrium protein folding intermediates [Ermácora, M. R., Delfino, J. M., Cuenoud, B., Schepartz, A., & Fox, R. O. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 6383-6387]. The chemical structure of protein cleavage products and the mechanism of backbone scission for this class of reagents have been unclear. Here, we report the nature of EPD-Fe-mediated backbone cleavage of a small model peptide. The EPD-Fe reagent was attached to a partially alpha-helical peptide, alpha 1BA1a (Ac-AEAEEAAKKAKEACKA-NH2), through a mixed disulfide. Backbone cleavage was initiated by addition of the iron reductant ascorbate. Chemical analysis of the novel cleavage products revealed an oxidative cleavage mechanism, probably initiated by diffusible hydroxyl radicals. The EPD-Fe-mediated cleavage technique appears to be suitable for the analysis of nonnative protein states such as the molten globule.

Single cysteine-substituted mutants of gamma delta resolvase have been covalently modified using a novel sulfhydryl-specific EDTA derivative, EDTA-2-aminoethyl 2-pyridyl disulfide (EPD). Iron, chelated by the coupled EDTA and in the presence of reducing agent, generates reactive oxygen species that result in localized cleavage of the DNA to which resolvase is bound. The procedure provides valuable information on two fronts. First, it allows the identification of regions or surfaces of the protein that are in close proximity to DNA even though they may not be part of the DNA-binding domain. Second, it allows identification of the portions of DNA that are closest to each EDTA-derivatized cysteine, since the DNA cleavages observed are highly localized and their efficiency drops rapidly as a function of the distance between the EDTA-Fe complex and the deoxyribose target. We have used the procedure to investigate the interaction of gamma delta resolvase with the three DNA binding sites that constitute its recombination substrate, res. The data indicate that the two N-terminal domains of a resolvase dimer interact symmetrically with site I, which contains the recombination cross-over point, but asymmetrically with the accessory sites, II and III. The patterns of DNA cleavage obtained with several different EDTA-coupled mutants have enabled us to propose a model for the interaction between resolvase and site I.

IA-2 (insulinoma-associated protein 2) is a protein-tyrosine phosphatase receptor located in secretory granules of neuroendocrine cells. Initially, it attracted attention due to its involvement in the autoimmune response associated to diabetes. Later it was found that upon exocytosis, the cytoplasmic domain of IA-2 is cleaved and relocated to the nucleus, where it enhances the transcription of the insulin gene. A concerted functioning of the whole receptor is to be expected. However, very little is known about the structure and function of the transmembrane and extracellular domains of IA-2. To address this issue, we solved the x-ray structure of the mature ectodomain of IA-2 (meIA-2) to 1.30 Å resolution. The fold of meIA-2 is related to the SEA (sea urchin sperm protein, enterokinase, agrin)) domains of mucins, suggesting its participation in adhesive contacts to the extracellular matrix and providing clues on how this kind of molecule may associate and form homo-and heterodimers. Moreover, we discovered that meIA-2 is self-proteolyzed in vitro by reactive oxygen species, suggesting the possibility of a new shedding mechanism that might be significant in normal function or pathological processes. Knowledge of meIA-2 structure should facilitate the search of its possible ligands and molecular interactions. Protein-tyrosine phosphatases (PTP),2 together with the corresponding kinases, regulate cell division, growth, differentiation, and metabolism (1). There are cytoplasmic PTP as well as transmembrane receptors PTP (RPTP). The latter also participate in cell-cell and cell-matrix contacts, possess an impressive diversity of adhesive and multimerization modules (2), and have been involved in human diseases such as cancer, autoimmunity, and degenerative processes (1).Two paralog RPTPs, IA-2 (insulinoma-associated protein 2, also termed PTP35 or ICA512) and IA-2 ␤ (PTPR2, also known as phogrin or IAR), were identified as major autoantigens in type-1 diabetes mellitus (3). They have a signal peptide, an ectodomain, a single-pass transmembrane region, and a single intracellular PTP domain.IA-2 and IA-2 ␤ are prominent in the secretory granules (SG) of brain, pituitary, pancreatic islet, and adrenal endocrine cells (4). Although the physiological ligands for these receptors are unknown and their function is poorly understood, they are involved in hormone and neuropeptide secretion. Indeed, single-and double-knock-out mice lacking IA-2 suffer from glucose intolerance, impaired insulin secretion, and abnormal secretion of pituitary hormones and female infertility (5, 6).Processing of pro IA-2 by furin-like hormone convertases produces mature IA-2 (7), which lacks the signal peptide and an adjacent fragment (residues 1-448). Mature IA-2 reaches the plasma membrane during exocytosis and comprises extracellular (449 -575), transmembrane (576 -600), and cytoplasmic domains (601-979). High glucose levels up-regulate IA-2 (8), and insulin exocytosis triggers a Ca 2ϩ -dependent and -calpain-mediated cleavage of the IA-2 cytopl...

Autoantibodies to the islet-cell 65-kDa variant of glutamate decarboxylase (GAD65) are found in most insulin-dependent diabetes mellitus (IDDM) patients many years before the appearance of clinical symptoms of the disease. As IDDM-preventive therapies may be available in the future, an international effort is taking place to develop widely applicable anti-GAD immunochemical tests. These tests would help to detect individuals at risk before the full installation of the disease and to enroll them in prevention programs. Autoantibodies to GAD65 are mostly directed to conformational epitopes, and the enzyme is a complex molecule with a prosthetic group and 15 cysteine residues. Thus, the conformational integrity of GAD65 is essential for an appropriate anti-GAD assay. Isolation of large amounts of GAD65 from pancreas or other tissues is impractical, and no successful production of properly folded GAD65 has been reported in bacteria. Native recombinant GAD65 for immunochemical tests is usually obtained from eukaryotic expression systems. Since the large-scale production of a recombinant protein in an eukaryotic system is expensive and technically difficult, we investigated the expression of GAD65 in Escherichia coli as an alternative. A number of DNA constructs intended to export the enzyme to the periplasmic space or to improve its cytoplasmic solubility were designed and tested. Our results provide a solution to the two main problems associated with the expression of GAD65 in E. coli: misfolding, leading to the formation of inclusion bodies ; and the presence of alternative initiation sites for translation that causes the preferential production of truncated variants of GAD65. We describe here the production of properly folded, fully active, and immunochemically competent GAD65 as an N-terminal fusion protein with thioredoxin. An account of the reactivity of the produced protein with sera of six IDDM patients is also presented.

Searching for bioactive peptides, we analyzed acidic extracts of Phyllomedusa sauvagii skin and found two new proteins, PSKP-1 and PSKP-2, of 6.7 and 6.6 kDa, respectively, which, by sequence homology, belong to the Kazal family of serine protease inhibitors. PSKP-1 and PSKP-2 exhibit the unprecedented feature of having proline at P 1 and P 2 positions. A gene encoding PSKP-1 was synthesized and expressed in Escherichia coli. Recombinant PSKP-1 was purified from inclusion bodies, oxidatively refolded to the native state, and characterized by chemical, hydrodynamic and optical studies. PSKP-1 shows inhibitory activity against a serum prolyl endopeptidase, but is unable to inhibit trypsin, chymotrypsin, V8 protease, or proteinase K. In addition, PSKP-1 can be rendered active against trypsin by activesite site-specific mutagenesis, has bactericidal activity, and induces agglutination of red cells at micromolar concentrations. PSKP-1 might protect P. sauvagii teguments from microbial invasion, by acting as an inhibitor of an as-yet unidentified prolyl endopeptidase or directly as a microbicidal compound.

Non-native states of proteins populated at extremes of pH, or by mutation or truncation of the protein sequence, are thought to be equilibrium models for kinetic intermediates on the folding pathway. While the global physical properties of these molecules have been well characterized, analysis of their structure by NMR spectroscopy has proven difficult. Here we report the use of a new chemical cleavage technique, based on reactive oxygen species, to map the backbone fold of a truncated form of staphylococcal nuclease in a non-native state. The fragment adopts a native-like fold, however the technique also reveals regions of non-native structure.

Two genetically engineered variants of the Bacillus licheniformis b-lactamase gene were expressed in Escherichia coli. One variant coded for the exo-small mature enzyme without the signal peptide. The other coded for the exo-large mature enzyme preceded by 10, mostly polar, residues from an incomplete heterologous signal. As observed following the extraction by a lysozyme-EDTA treatment, the signal-less variant was exported to the periplasm with nearly 20% efficiency, whereas the variant with the N-terminal extension was translocated to a lesser degree; interestingly, nearly all of the former and half of the latter were extracted by osmotic shock, which may be of importance for our understanding of cellular compartments. The fact that a signal-less protein is translocated with substantial yields raises questions about the essential role of signal peptides for protein export. As folding and export are related processes, we investigated the folding in vitro of the two variants. No differences were found between them. In the absence of denaturant, they are completely folded, fully active and have a large DG of unfolding. Under partially denaturing conditions they populate several partially folded states. The absence of significant amounts of a non-native state under native conditions makes a thermodynamic partitioning between folding and export less likely. In addition, kinetic measurements indicated that these B. licheniformis lactamases fold much faster than E. coli b-lactamase. This behavior suggests that they are exported by a kinetically controlled process, mediated by one or more still unidentified interactions that slow folding and allow a folding intermediate to enter the export pathway.Keywords: b-lactamase; protein export; protein folding; signal peptide.The information to guide protein folding and export is encoded by the amino acid sequence. As it is generally accepted that native structures do not cross membranes, it follows that protein folding must be coordinately regulated to allow protein export [1±4]. An additional complication is the potential for folding proteins to undergo aggregation, which could interfere with the kinetics of both folding and export.In some cases, folding is impeded cotranslationally, by attaching the functional ribosome to a membrane, sequestering the nascent peptide from the cytoplasm, and extruding it through a proteinaceous pore. In other cases, a much more interesting mechanism takes place that keeps a completely synthesized polypeptide in a non-native, water-soluble, exportcompetent state until translocation is achieved. In this mechanism, the folding code must be temporarily overridden by the export code.Most exported proteins have a signal sequence whose proposed functions are: (a) to keep the precursor in an export-competent state; (b) to interact with specific components of the export machinery; and (c) to initiate protein secretion by inserting itself in the membrane bilayer [5]. After proper localization, the signal is usually removed by specific proteases, releas...