Formation and seeding of amyloid fibrils from wild-type hen lysozyme and a peptide fragment from the beta-domain.

Wild-type hen lysozyme has been converted from its soluble native state into highly organized amyloid fibrils. In order to achieve this conversion, conditions were chosen to promote partial unfolding of the native globular fold and included heating of low-pH solutions and addition of organic solvents. Two peptides derived from the beta-sheet region of hen lysozyme were also found to form fibrils very readily. The properties and morphologies of the amyloid fibrils formed by incubation either of the protein or the peptides are similar to those produced from the group of proteins associated with clinical amyloidoses. Fibril formation by hen lysozyme was substantially accelerated when aliquots of solutions in which fibrils of either one of the peptides or the full-length protein had previously formed were added to fresh solutions of the protein, revealing the importance of seeding in the kinetics of fibril formation. These findings support the proposition that the beta-domain is of particular significance in the formation of fibrils from the full-length protein and suggest similarities between the species giving rise to fibril formation and the intermediates formed during protein folding.

Probing the nature of interactions in SH2 binding interfaces--evidence from electrospray ionization mass spectrometry.

We have adopted nanoflow electrospray ionization mass spectrometry (ESI-MS) and isothermal titration calorimetry (ITC) to probe the mechanism of peptide recognition by the SH2 domain from the Src family tyrosine kinase protein, Fyn. This domain is involved in the mediation of intracellular signal transduction pathways by interaction with proteins containing phosphorylated tyrosine (Y*) residues. The binding of tyrosyl phosphopeptides can mimic these interactions. Specificity in these interactions has been attributed to the interaction of the Y* and residues proximal and C-terminal to it. Previous studies have established that for specific binding with Fyn, the recognition sequence consists of pTyr-Glu-Glu-Ile. The specific interactions involve the binding of Y* with the ionic, and the Y* + 3 Ile residue with the hydrophobic binding pockets on the surface of the Fyn SH2 domain. In this work, a variation in the Y* + 3 residue of this high-affinity sequence was observed to result in changes in the relative binding affinities as determined in solution (ITC) and in the gas phase (nanoflow ESI-MS). X-ray analysis shows that a feature of the Src family SH2 domains is the involvement of water molecules in the peptide binding site. Under the nanoflow ESI conditions, water molecules appear to be maintained in the Fyn SH2-ligand complex. Compelling evidence for these molecules being incorporated in the SH2-peptide interface is provided by the prevalence of the peaks assigned to water-bound over the water-free complex at high-energy conditions. Thus, the stability of water protein-ligand complex appears to be intimately linked to the presence of water.

Formation of amyloid fibrils by peptides derived from the bacterial cold shock protein CspB.

Three peptides covering the sequence regions corresponding to the first two (CspB-1), the first three (CspB-2), and the last two (CspB-3) beta-strands of CspB, the major cold shock protein of Bacillus subtilis, have been synthesized and analyzed for their conformations in solution and for their precipitation behavior. The peptides are nearly insoluble in water, but highly soluble in aqueous solutions containing 50% acetonitrile (pH 4.0). Upon shifts of the solvent condition toward lower or higher acetonitrile concentrations, the peptides all form fibrils resembling those observed in amyloid associated diseases. These fibrils have been identified and characterized by electron microscopy, binding of the dye congo red, and X-ray fiber diffraction. Characterization of the peptides in solution by circular dichroism and NMR spectroscopy shows that the formation of these fibrils does not require specific preformed secondary structure in the solution state species. While the majority of the soluble fraction of each peptide is monomeric and unstructured, different types of structures including alpha-helical, beta-sheet, and random coil conformations are observed under conditions that eventually lead to fibril formation. We conclude that the absence of tertiary contacts under solution conditions where binding interactions between peptide units are still favorable is a crucial requirement for amyloid formation. Thus, fragmentation of a sequence, like partial chemical denaturation or mutation, can enhance the capacity of specific protein sequences to form such fibrils.