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) serve as a first line of defense against stress-induced cell damage by binding and maintaining denaturing proteins in a folding-competent state. In contrast to the well-defined substrate binding regions of ATP-dependent chaperones, interactions between sHSPs and substrates are poorly understood. Defining substrate-binding sites of sHSPs is key to understanding their cellular functions and to harnessing their aggregation-prevention properties for controlling damage due to stress and disease. We incorporated a photoactivatable cross-linker at 32 positions throughout a well-characterized sHSP, dodecameric PsHsp18.1 from pea, and identified direct interaction sites between sHSPs and substrates. Model substrates firefly luciferase and malate dehydrogenase form strong contacts with multiple residues in the sHSP N-terminal arm, demonstrating the importance of this flexible and evolutionary variable region in substrate binding. Within the conserved ␣-crystallin domain both substrates also bind the ␤-strand (␤7) where mutations in human homologs result in inherited disease. Notably, these binding sites are poorly accessible in the sHSP atomic structure, consistent with major structural rearrangements being required for substrate binding. Detectable differences in the pattern of cross-linking intensity of the two substrates and the fact that substrates make contacts throughout the sHSP indicate that there is not a discrete substrate binding surface. Our results support a model in which the intrinsicallydisordered N-terminal arm can present diverse geometries of interaction sites, which is likely critical for the ability of sHSPs to protect efficiently many different substrates.alpha-crystallin ͉ cross-linking ͉ intrinsic disorder ͉ P-benzoylphenylalanine ͉ protein-protein interactions

SUMMARY Small Heat-Shock Proteins (sHSPs) are a family of molecular chaperones that help prevent irreversible aggregation through binding non-native target proteins to form large and heterogeneous complexes. These sHSP:target complexes are an integral part of the overall proteostasis network but, due to their polydispersity, their composition and assembly remain poorly understood. Here, we present a novel nanoelectrospray mass spectrometry analysis algorithm for estimating the distribution of stoichiometries comprising a polydisperse ensemble of oligomers. We apply our approach to elucidate the organization of complexes formed between sHSPs and different target proteins. We find that protection of targets is mass-dependent, with the resultant complexes nonetheless reflecting properties of the target. Strikingly, we observe that aspects of the native quaternary architecture of the targets are retained, indicating that protection happens early in the denaturation process. Our data therefore explain the apparent paradox of how variable complex morphologies result from the generic mechanism of target protection afforded by the sHSPs. Our approach is applicable to a wide range of polydisperse proteins, and, more generally, provides a means for the automated and accurate interpretation of mass spectra derived from heterogeneous protein assemblies.

The dynamics of protein complexes are crucial for their function yet are challenging to study. Here, we present a nanoelectrospray (nESI) mass spectrometry (MS) approach capable of simultaneously providing structural and dynamical information for protein complexes. We investigate the properties of two small heat shock proteins (sHSPs) and find that these proteins exist as dodecamers composed of dimeric building blocks. Moreover, we show that these proteins exchange dimers on the timescale of minutes, with the rate of exchange being strongly temperature dependent. Because these proteins are expressed in the same cellular compartment, we anticipate that this dynamical behavior is crucial to their function in vivo. Furthermore, we propose that the approach used here is applicable to a range of nonequilibrium systems and is capable of providing both structural and dynamical information necessary for functional genomics.

Interpretation of deglycosylation studies relies heavily on the absence of modifications to the polypeptide chain. We have found that by using a common chemical deglycosylation technique, one can effect at least three changes in a peptide's structure: methylation, isomerization, and ring formation. It was determined that the conditions of chemical deglycosylation introduce a +14 Da shift in the masses of our model peptides, RKDVY, RKEVY, and horseradish peroxidase. This shift is localized to acidic functional groups and is interpreted as methylation of the free carboxylates in our models. An additional shift in mass of -18 Da is found in the model peptide RKDVY consistent with the loss of water associated with succinimide ring formation in this peptide. Chemical treatment induced isomerization of aspartyl residues to isoaspartyl residues in another model peptide, tetragastrin. These results indicate that one should use caution when interpreting the results of chemical deglycosylation experiments.