The small heat shock proteins (sHSPs) and the related α-crystallins (αCs) are virtually ubiquitous proteins that are strongly induced by a variety of stresses, but that also function constitutively in multiple cell types in many organisms. Extensive research demonstrates that a majority of sHSPs and αCs can act as ATP-independent molecular chaperones by binding denaturing proteins and thereby protecting cells from damage due to irreversible protein aggregation. Because of their diverse evolutionary history, their connection to inherited human diseases, and their novel protein dynamics, sHSPs and αCs are of significant interest to many areas of biology and biochemistry. However, it is increasingly clear that no single model is sufficient to describe the structure, function or mechanism of action of sHSPs and αCs. In this review, we discuss recent data that provide insight into of the variety of structures of these proteins, their dynamic behavior, how they recognize substrates, and their many possible cellular roles.

The 2.7 A structure of wheat HSP16.9, a member of the small heat shock proteins (sHSPs), indicates how its alpha-crystallin domain and flanking extensions assemble into a dodecameric double disk. The folding of the monomer and assembly of the oligomer are mutually interdependent, involving strand exchange, helix swapping, loose knots and hinged extensions. In support of the chaperone mechanism, the substrate-bound dimers, in temperature-dependent equilibrium with higher assembly forms, have unfolded N-terminal arms and exposed conserved hydrophobic binding sites on the alpha-crystallin domain. The structure also provides a model by which members of the sHSP protein family bind unfolded substrates, which are involved in a variety of neurodegenerative diseases and cataract formation.

Small Heat Shock Proteins (sHSPs) are a diverse family of molecular chaperones that prevent protein aggregation by binding clients destabilized during cellular stress. Here we probe the architecture and dynamics of complexes formed between an oligomeric sHSP and client by employing unique mass spectrometry strategies. We observe over 300 different stoichiometries of interaction, demonstrating that an ensemble of structures underlies the protection these chaperones confer to unfolding clients. This astonishing heterogeneity not only makes the system quite distinct in behavior to ATP-dependent chaperones, but also renders it intractable by conventional structural biology approaches. We find that thermally regulated quaternary dynamics of the sHSP establish and maintain the plasticity of the system. This extends the paradigm that intrinsic dynamics are crucial to protein function to include equilibrium fluctuations in quaternary structure, and suggests they are integral to the sHSPs' role in the cellular protein homeostasis network.heterogeneity | mass spectrometry | polydispersity | protein dynamics | proteostasis S mall Heat Shock Proteins (sHSPs) are one of the least well understood classes of molecular chaperones, proteins which act to prevent or reverse improper protein associations (1). The importance of the sHSPs is evidenced by their almost ubiquitous expression (2), the presence of multiple sHSP genes in most organisms (3), and their dramatic up-regulation under stress conditions making them among the most abundant of cellular proteins (4). They are implicated in a range of disease states including cataract, cancer, myopathies, motor neuropathies, and neurodegeneration (5-8). The current view of their chaperone action is that they bind unfolding "client" proteins, thereby preventing their irreversible aggregation (9-12). These sHSP: client complexes then interact with ATP-dependent chaperones to allow refolding of the clients (9-12). Structural interrogation of the complexes they form with clients has however been hampered by their apparent heterogeneity, and their organization remains consequently very poorly defined (13-15).MS is an emergent technology for the structural biology of protein assemblies (16), allowing the interrogation of a wide range of biomolecular systems, including those complicated by polydispersity and dynamics (17, 18). Here we capitalize on these unique advantages to study the complexes formed between pea HSP18.1 and a model client protein, firefly luciferase (Luc). HSP18.1 represents the family of class I cytosolic plant sHSPs which accumulate at heat-shock temperatures (≥38°C) to ≈1% of the total cellular protein (19). Extensive in vitro studies have established that HSP18.1 is able to bind destabilized clients, enabling subsequent refolding by the HSP70 machinery (20,21). With the in vivo clients of HSP18.1 yet to be identified, Luc was chosen as it is extremely thermo-sensitive and has been used extensively in chaperone studies (12). Luc does not interact with HSP18.1 at room te...

Small heat shock proteins (sHSPs) are a ubiquitous class of molecular chaperones that interacts with substrates to prevent their irreversible insolubilization during denaturation. How sHSPs interact with substrates remains poorly defined. To investigate the role of the conserved C-terminal ␣-crystallin domain versus the variable N-terminal arm in substrate interactions, we compared two closely related dodecameric plant sHSPs, Hsp18.1 and Hsp16.9, and four chimeras of these two sHSPs, in which all or part of the N-terminal arm was switched. The efficiency of substrate protection and formation of sHSPsubstrate complexes by these sHSPs with three different model substrates, firefly luciferase, citrate synthase, and malate dehydrogenase (MDH) provide new insights into sHSP/substrate interactions. Results indicate that different substrates have varying affinities for different domains of the sHSP. For luciferase and citrate synthase, the efficiency of substrate protection was determined by the identity of the N-terminal arm in the chimeric proteins. In contrast, for MDH, efficient protection clearly required interactions with the ␣-crystallin domain in addition to the N-terminal arm. Furthermore, we show that sHSP-substrate complexes with varying stability and composition can protect substrate equally, and substrate protection is not correlated with sHSP oligomeric stability for all substrates. Protection of MDH by the dimeric chimera composed of the Hsp16.9 N-terminal arm and Hsp18.1 ␣-crystallin domain supports the model that a dimeric form of the sHSP can bind and protect substrate. In total, results demonstrate that sHSP-substrate interactions are complex, likely involve multiple sites on the sHSP, and vary depending on substrate.The small heat shock proteins (sHSPs), 4 and the related ␣-crystallins comprise a superfamily of chaperones defined by a conserved C-terminal domain of ϳ90 amino acids referred to as the ␣-crystallin domain (1). Flanking this domain is a short C-terminal extension and an N-terminal arm of variable length and highly divergent sequence (1-3). Although the monomeric size of the sHSPs ranges from 15 to 42 kDa, in their native state most sHSPs assemble into large oligomers of 8 to Ͼ32 subunits, although there are also dimeric and tetrameric sHSPs (1, 4). 5Studies with numerous sHSPs from different organisms have shown that in vitro these proteins act as chaperones by binding to partially unfolded proteins in an ATP-independent manner, preventing their irreversible aggregation (1, 5). Substrates that are denatured in the presence of sHSPs can then be refolded and reactivated by the ATP-dependent chaperone DnaK/ Hsp70 with the participation in some cases of ClpB/Hsp100 and GroEL (1, 6, 7). In vivo, increased expression of these ubiquitous stress proteins can enhance cellular tolerance to a variety of stresses, such as heat, salt, drugs, and oxidants (1). sHSPs have also been reported to act as negative regulators of apoptosis, to modulate cellular redox state, and to be linked to increased orga...

The small heat shock proteins (sHSPs) are a ubiquitous class of ATP-independent chaperones believed to prevent irreversible protein aggregation and to facilitate subsequent protein renaturation in cooperation with ATP-dependent chaperones. Although sHSP chaperone activity has been studied extensively in vitro, understanding the mechanism of sHSP function requires identification of proteins that are sHSP substrates in vivo. We have used both immunoprecipitation and affinity chromatography to recover 42 proteins that specifically interact with Synechocystis Hsp16.6 in vivo during heat treatment. These proteins can all be released from Hsp16.6 by the ATP-dependent activity of DnaK and cochaperones and are heat-labile. Thirteen of the putative substrate proteins were identified by mass spectrometry and reveal the potential for sHSPs to protect cellular functions as diverse as transcription, translation, cell signaling, and secondary metabolism. One of the putative substrates, serine esterase, was purified and tested directly for interaction with purified Hsp16.6. Hsp16.6 effectively formed soluble complexes with serine esterase in a heat-dependent fashion, thereby preventing formation of insoluble serine esterase aggregates. These data offer critical insights into the characteristics of native sHSP substrates and extend and provide in vivo support for the chaperone model of sHSP function.The small heat shock proteins (sHSPs) 1 and the structurally related vertebrate eye lens ␣-crystallins are a family of virtually ubiquitous stress proteins (1, 2). Their role in cellular stress extends from protection against high temperature and oxidative stress to a potentially important function in a variety of protein-folding diseases and aging (3, 4). The sHSPs are defined by a conserved C-terminal domain of ϳ90 amino acids (the ␣-crystallin domain), which is flanked by a variable length N-terminal arm and a more conserved C-terminal extension (1, 5). These small proteins (16 -42-kDa monomers) assemble into oligomeric structures of 9 to Ͼ32 subunits depending on the sHSP (6, 7). sHSPs are very efficient at binding denatured proteins, and current models propose that they function as ATP-independent molecular chaperones to prevent irreversible protein aggregation and insolubilization (2). sHSP-bound proteins can be reactivated by the ATP-dependent chaperone activity of DnaK/Hsp70, with the help of ClpB or GroEL in some instances (8 -10). Thus, sHSPs are proposed to be a critical component of the cellular chaperone network that becomes particularly important under conditions of severe stress involving protein aggregation.Although the ability of sHSPs to interact with denatured model substrates in vitro has been studied extensively, the characteristics of cellular sHSP substrates remain poorly defined. Understanding the mechanism of sHSP chaperone action, as well as determining how these proteins may act to protect cells during stress, requires identification of proteins that interact with sHSPs in vivo, either as partners or subst...