Multiple distinct assemblies reveal conformational flexibility in the small heat shock protein Hsp26.

Small heat shock proteins are a superfamily of molecular chaperones that suppress protein aggregation and provide protection from cell stress. A key issue for understanding their action is to define the interactions of subunit domains in these oligomeric assemblies. Cryo-electron microscopy of yeast Hsp26 reveals two distinct forms, each comprising 24 subunits arranged in a porous shell with tetrahedral symmetry. The subunits form elongated, asymmetric dimers that assemble via trimeric contacts. Modifications of both termini cause rearrangements that yield a further four assemblies. Each subunit contains an N-terminal region, a globular middle domain, the alpha-crystallin domain, and a C-terminal tail. Twelve of the C termini form 3-fold assembly contacts which are inserted into the interior of the shell, while the other 12 C termini form contacts on the surface. Hinge points between the domains allow a variety of assembly contacts, providing the flexibility required for formation of supercomplexes with non-native proteins.

Spider silk and amyloid fibrils: a structural comparison.

Although spider silks have been studied for decades, the assembly properties of the underlying silk proteins have still not been unravelled. Previously, the detection of amyloid-like nanofibrils in the spider's silk gland suggested their involvement in the assembly process.Recombinantly produced spider silk also self-assembles into nanofibrils. In order to investigate the structural properties of such silk nanofibrils in more detail, they have been compared to amyloid-like fibrils to highlight structural similarities.

Insights into the architecture of the Ure2p yeast protein assemblies from helical twisted fibrils.

The protein Ure2 from baker's yeast is associated with a heritable and transmissible phenotypic change in the yeast Saccharomyces cerevisiae. Such prion properties are thought to arise from the fact that Ure2p is able to self-assemble into insoluble fibrils. Assemblies of Ure2p are composed of full-length proteins in which the structure of the globular, functional, C-terminal domain is retained. We have carried out structural studies on full-length, wild-type Ure2p fibrils with a regularly twisted morphology. Using electron microscopy and cryo-electron microscopy with image analysis we show high-resolution images of the twisted filaments revealing details within the fibrillar structure. We examine these details in light of recent proposed models and discuss how this new information contributes to an understanding of the architecture of Ure2p yeast prion fibrils.

A domain in the N-terminal part of Hsp26 is essential for chaperone function and oligomerization.

Small heat-shock proteins (Hsps) are ubiquitous molecular chaperones which prevent the unspecific aggregation of non-native proteins. For Hsp26, a cytosolic sHsp from of Saccharomyces cerevisiae, it has been shown that, at elevated temperatures, the 24 subunit complex dissociates into dimers. This dissociation is required for the efficient interaction with non-native proteins. Deletion analysis of the protein showed that the N-terminal half of Hsp26 (amino acid residues 1-95) is required for the assembly of the oligomer. Limited proteolysis in combination with mass spectrometry suggested that this region can be divided in two parts, an N-terminal segment including amino acid residues 1-30 and a second part ranging from residues 31-95. To analyze the structure and function of the N-terminal part of Hsp26 we created a deletion mutant lacking amino acid residues 1-30. We show that the oligomeric state and the structure, as determined by size exclusion chromatography and electron microscopy, corresponds to that of the Hsp26 wild-type protein. Furthermore, this truncated version of Hsp26 is active as a chaperone. However, in contrast to full length Hsp26, the truncated version dissociates at lower temperatures and complexes with non-native proteins are less stable than those found with wild-type Hsp26. Our results suggest that the N-terminal segment of Hsp26 is involved in both, oligomerization and chaperone function and that the second part of the N-terminal region (amino acid residues 31-95) is essential for both functions.

Structure and morphology of the Alzheimer's amyloid fibril.

Amyloid fibrils are deposited in a number of diseases, including Alzheimer's disease, Type 2 diabetes, and the transmissible spongiform encephalopathies (TSE). These insoluble deposits are formed from normally soluble proteins that assemble to form fibrous aggregates that accumulate in the tissues. Electron microscopy has been used as a tool to examine the structure and morphology of these aggregates from ex vivo materials, but predominantly from synthetic amyloid fibrils assembled from proteins or peptides in vitro. Electron microscopy has shown that the fibrils are straight, unbranching, and are of a similar diameter (60-100 A) irrespective of the precursor protein. Image processing has enhanced electron micrographs to show that amyloid fibrils appear to be composed of protofilaments wound around one another. In combination with other techniques, including X-ray fiber diffraction and solid state NMR, electron microscopy has revealed that the internal structure of the amyloid fibril is a ladder of beta-sheet structure arranged in a cross-beta conformation.

Atomic models of de novo designed cc beta-Met amyloid-like fibrils.

The common characteristics of amyloid and amyloid-like fibrils from disease- and non-disease-associated proteins offer the prospect that well-defined model systems can be used to systematically dissect the driving forces of amyloid formation. We recently reported the de novo designed cc beta peptide model system that forms a native-like coiled-coil structure at low temperatures and which can be switched to amyloid-like fibrils by increasing the temperature. Here, we report a detailed molecular description of the system in its fibrillar state by characterizing the cc beta-Met variant using several microscopic techniques, circular dichroism spectroscopy, X-ray fiber diffraction, solid-state nuclear magnetic resonance, and molecular dynamics calculations. We show that cc beta-Met forms amyloid-like fibrils of different morphologies on both the macroscopic and atomic levels, which can be controlled by variations of assembly conditions. Interestingly, heterogeneity is also observed along single fibrils. We propose atomic models of the cc beta-Met amyloid-like fibril, which are in good agreement with all experimental data. The models provide a rational explanation why oxidation of methionine residues completely abolishes cc beta-Met amyloid fibril formation, indicating that a small number of site-specific hydrophobic interactions can play a major role in the packing of polypeptide-chain segments within amyloid fibrils. The detailed structural information available for the cc beta model system provides a strong molecular basis for understanding the influence and relative contribution of hydrophobic interactions on native-state stability, kinetics of fibril formation, fibril packing, and polymorphism.

Disassembling protein aggregates in the yeast cytosol. The cooperation of Hsp26 with Ssa1 and Hsp104.

In all organisms studied, elevated temperatures induce the expression of a variety of stress proteins, among them small Hsps (sHsp). sHsps are chaperones that prevent the unspecific aggregation of proteins by forming stable complexes with unfolded polypeptides. Reactivation of captured proteins requires the assistance of other ATP-dependent chaperones. How sHsps and ATP-dependent chaperones work together is poorly understood. Here, we analyzed the interplay of chaperones present in the cytosol of Saccharomyces cerevisiae. Specifically, we characterized the influence of Hsp104 and Ssa1 on the disassembly of Hsp26 x substrate complexes in vitro and in vivo. We show that recovery of proteins from aggregates in the cell requires the chaperones to work together with defined but overlapping functions. During reactivation, proteins are transferred from a stable complex with Hsp26 to Hsp104 and Hsp70. The need for ATP-dependent chaperones depends on the type of sHsp x substrate complex. Although Ssa1 is able to release substrate proteins from soluble Hsp26 x substrate complexes, Hsp104 is essential to dissociate substrate proteins from aggregates with incorporated sHsps. Our results are consistent with a model of several interrelated defense lines against protein aggregation.

Analysis of the interaction of small heat shock proteins with unfolding proteins.

The ubiquitous small heat shock proteins (sHsps) are efficient molecular chaperones that interact with nonnative proteins, prevent their aggregation, and support subsequent refolding. No obvious substrate specificity has been detected so far. A striking feature of sHsps is that they form large complexes with nonnative proteins. Here, we used several well established model chaperone substrates, including citrate synthase, alpha-glucosidase, rhodanese, and insulin, and analyzed their interaction with murine Hsp25 and yeast Hsp26 upon thermal unfolding. The two sHsps differ in their modes of activation. In contrast to Hsp25, Hsp26 undergoes a temperature-dependent dissociation that is required for efficient substrate binding. Our analysis shows that Hsp25 and Hsp26 reacted in a similar manner with the nonnative proteins. For all substrates investigated, complexes of defined size and shape were formed. Interestingly, several different nonnative proteins could be incorporated into defined sHsp-substrate complexes. The first substrate protein bound seems to determine the complex morphology. Thus, despite the differences in quaternary structure and mode of activation, the formation of large uniform sHsp-substrate complexes seems to be a general feature of sHsps, and this unique chaperone mechanism is conserved from yeast to mammals.

Hsp42 is the general small heat shock protein in the cytosol of Saccharomyces cerevisiae.

Small heat shock proteins (sHsps) are ubiquitous molecular chaperones that prevent the unspecific aggregation of proteins. So far, Hsp26 was the only unambiguously identified member of the sHsp family in Saccharomyces cerevisiae. We show here that the sHsp system in the cytosol of S. cerevisiae consists of two proteins, Hsp26 and Hsp42. Hsp42 forms large dynamic oligomers with a barrel-like structure. In contrast to Hsp26, which functions predominantly at heat shock temperatures, Hsp42 is active as a chaperone under all conditions tested in vivo and in vitro. Under heat shock conditions, both Hsp42 and Hsp26 suppress the aggregation of one-third of the cytosolic proteins. This subset is about 90% overlapping for Hsp42 and Hsp26. The sHsp substrates belong to different biochemical pathways. This indicates a general protective function of sHsps for proteome stability in S. cerevisiae. Consistent with this observation, sHsp knockout strains show phenotypical defects. Taken together, our results define Hsp42 as an important player for protein homeostasis at physiological and under stress conditions.

Analysis of the regulation of the molecular chaperone Hsp26 by temperature-induced dissociation: the N-terminal domail is important for oligomer assembly and the binding of unfolding proteins.

Small heat shock proteins (sHsps) are molecular chaperones that efficiently bind non-native proteins. All members of this family investigated so far are oligomeric complexes. For Hsp26, an sHsp from the cytosol of Saccharomyces cerevisiae, it has been shown that at elevated temperatures the 24-subunit complex dissociates into dimers. This dissociation seems to be required for the efficient interaction with unfolding proteins that results in the formation of large, regular complexes comprising Hsp26 and the non-native proteins. To gain insight into the molecular mechanism of this chaperone, we analyzed the dynamics and stability of the two oligomeric forms of Hsp 26 (i.e. the 24-mer and the dimer) in comparison to a construct lacking the N-terminal domain (Hsp26DeltaN). Furthermore, we determined the stabilities of complexes between Hsp26 and non-native proteins. We show that the temperature-induced dissociation of Hsp26 into dimers is a completely reversible process that involves only a small change in energy. The unfolding of the dissociated Hsp26 dimer or Hsp26DeltaN, which is a dimer, requires a much higher energy. Because Hsp26DeltaN was inactive as a chaperone, these results imply that the N-terminal domain is of critical importance for both the association of Hsp26 with non-native proteins and the formation of large oligomeric complexes. Interestingly, complexes of Hsp26 with non-native proteins are significantly stabilized against dissociation compared with Hsp26 complexes. Taken together, our findings suggest that the quaternary structure of Hsp26 is determined by two elements, (i) weak, regulatory interactions required to form the shell of 24 subunits and (ii) a strong and stable dimerization of the C-terminal domain.