Native zinc-bound insulin exists as a hexamer at neutral pH. Under destabilizing conditions, the hexamer dissociates, and is very prone to forming fibrils. Insulin fibrils exhibit the typical properties of amyloid fibrils, and pose a problem in the purification, storage, and delivery of therapeutic insulin solutions. We have carried out a systematic investigation of the effect of guanidine hydrochloride (Gdn.HCl)-induced structural perturbations on the mechanism of fibrillation of insulin. At pH 7.4, the addition of as little as 0.25 M Gdn.HCl leads to dissociation of insulin hexamers into dimers. Moderate concentrations of Gdn.HCl lead to formation of a novel partially unfolded dimer state, which dissociates into a partially unfolded monomer state. High concentrations of Gdn.HCl resulted in unfolded monomers with some residual structure. The addition of even very low concentrations of Gdn.HCl resulted in substantially accelerated fibrillation, although the yield of fibrils decreased at high concentrations. Accelerated fibrillation correlated with the population of the expanded (partially folded) monomer, which existed up to >6 M Gdn.HCl, accounting for the formation of substantial amounts of fibrils under such conditions. In the presence of 20% acetic acid, where insulin exists as the monomer, fibrillation was also accelerated by Gdn.HCl. The enhanced fibrillation of the monomer was due to the increased ionic strength at low denaturant concentrations, and due to the presence of the partially unfolded, expanded conformation at Gdn.HCl concentrations above 1 M. The data suggest that under physiological conditions, the fibrillation of insulin involves both changes in the association state (with rate-limiting hexamer dissociation) and conformational changes, leading to formation of the amyloidogenic expanded monomer intermediate.

Insulin has a largely ␣-helical structure and exists as a mixture of hexameric, dimeric, and monomeric states in solution, depending on the conditions: the protein is monomeric in 20% acetic acid. Insulin forms amyloid-like fibrils under a variety of conditions, especially at low pH. In this study we investigated the fibrillation of monomeric human insulin by monitoring changes in CD, attenuated total reflectance-Fourier transform infrared spectroscopy, 8-anilinonaphthalenesulfonic acid fluorescence, thioflavin T fluorescence, dynamic light scattering, and H/D exchange during the initial stages of the fibrillation process to provide insight into early events involving the monomer. The results demonstrate the existence of structural changes occurring before the onset of fibril formation, which are detectable by multiple probes. The data indicate at least two major populations of oligomeric intermediates between the native monomer and fibrils. Both have significantly non-native conformations, and indicate that fibrillation occurs from a betarich structure significantly distinct from the native fold.A number of human diseases are caused by the pathogenic deposition of proteins in the form of amyloid-like fibrils (1-7). Several non-pathogenic proteins and peptides also undergo amyloid like fibril formation on destabilization of their native state (7-10). The fact that structurally and sequentially non-homologous proteins are able to self-assemble into fibrils possessing similar morphology (e.g. 10 -18 nm width, birefringence to polarized light, and cross-␤ structure) suggests a common molecular mechanism in the fibrillation pathways. A variety of hypotheses for the mechanism of fibril formation have been proposed.Insulin is a 51-residue hormone with a largely ␣-helical structure. It exists as a mixture of hexameric, dimeric, and monomeric states in solution, with the relative population of different oligomeric species being strongly dependent on the environmental conditions: the protein is predominantly monomeric in 20% acetic acid, dimeric in 20 mM HCl, and hexameric at pH 7.5 in the presence of zinc. Insulin forms amyloidlike fibrils under a variety of conditions (11-13), with various overall morphologies depending on the arrangement of constituent protofilaments (14, 15). Insulin fibrils pose a variety of problems in biomedical and biotechnological applications. Amyloid deposits of insulin have been observed in patients with diabetes after repeated injection and in normal aging, as well as after subcutaneous insulin infusion (16,17).In our previous work we have shown the important role of partially folded intermediates in insulin fibrillation in vitro (13, 18 -20). In fact, our studies showed that insulin, which is a hexamer at physiological pH, undergoes rapid fibrillation starting at relatively low concentrations of guanidine hydrochloride and urea. The predominant species characterized by various biophysical techniques under these conditions was shown to be a partially folded, expanded monomer, which is presen...

Heat shock 70 kDa (Hsp70) chaperones are essential to in-vivo protein folding, protein transport and protein re-folding. They carry out these activities using repeated cycles of binding and release of client proteins. This process is under allosteric control of nucleotide binding and hydrolysis. X-ray crystallography, NMR spectroscopy and other biophysical techniques have contributed much to the understanding of the allosteric mechanism linking these activities and the effect of co-chaperones on this mechanism. In this chapter, these findings are critically reviewed. Studies on the allosteric mechanisms of Hsp70 have gained enhanced urgency, as recent studies have implicated this chaperone as a potential drug target in diseases such as Alzheimer's and cancer. Recent approaches to combat these diseases through interference with the Hsp70 allosteric mechanism are discussed.

The heat shock protein 70 kDa (Hsp70)/DnaJ/nucleotide exchange factor system assists in intracellular protein (re)folding. Using solution NMR, we obtained a three-dimensional structure for a 75-kDa Hsp70-DnaJ complex in the ADP state, loaded with substrate peptide. We establish that the J domain (residues 1-70) binds with its positively charged helix II to a negatively charged loop in the Hsp70 nucleotide-binding domain. The complex shows an unusual "tethered" binding mode which is stoichiometric and saturable, but which has a dynamic interface. The complex represents part of a triple complex of Hsp70 and DnaJ both bound to substrate protein.Mutagenesis data indicate that the interface is also of relevance for the interaction of Hsp70 and DnaJ in the ATP state. The solution complex is completely different from a crystal structure of a disulfide-linked complex of homologous proteins [Jiang, et al. (2007) Mol Cell 28:422-433].protein interactions | structural biology T he heat shock protein 70 kDa, heat shock protein 40 kDa, nucleotide exchange factor (Hsp70/Hsp40/NEF) system, is an essential chaperone system that facilitates the folding and refolding of proteins in healthy and stressed cells (1). The system is upregulated in tumors (2), is involved in Alzheimer's disease (3), and is an emerging target for therapy of these diseases (3). In this work, we study the bacterial Hsp70/Hsp40/NEF system, which is called DnaK/DnaJ/GrpE. Because DnaK and DnaJ are, respectively, 68% and 54% homologous to their human counterparts Hsc70 and HDJ2, the bacterial system is generally viewed as a prototype for the human Hsp70 chaperone system. DnaK is an allosteric protein, in which ATP binding at the nucleotide-binding domain (NBD) causes substrate release at the substrate-binding domain (SBD) with opening of the LID (residues 508-602) (1). Although structures for the individual domains and several truncations have long been known, only one structure for a near complete, not mutated Hsp70 is available to date: DnaK(1-605) of Escherichia coli in the presence of ADP, and the substrate peptide NRLLLTG (NR) (4). In this structure, the LID domain is docked to the SBD, but the SBD-LID unit moves rather unrestricted with respect to the NBD. The NBD and SBD of DnaK do interact in the ATP state (5, 6), but no structure for any Hsp70 in that state has been determined to date.E. coli DnaJ contains an N-terminal 70-residue J domain, followed by an approximately 40-residue Gly/Phe-rich (GF) region, a Zn-Cys domain, a substrate-binding domain, and a dimerization domain. DnaJ binds to stretches of exposed hydrophobic residues in client proteins (7). The J domain alone has been shown to be sufficient to stimulate ATPase activity of DnaK (8) Residues 1-70 form an antiparallel two-helix bundle, referred to as helices II and III, with two small adjacent helical elements (9, 10). The positively charged residues referred to above are in helix II, whereas the HPD motif is located in a loop connecting helices II and III. The GF region (residues 71-108) ...