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...
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.
The gas phase structures of phenyl alpha- and beta-d-xylopyranoside (alpha- and beta-pXyl) and their mono-hydrates have been investigated using a combination of resonant two-photon ionization (R2PI), ultra-violet hole-burning and resonant infrared ion dip spectroscopy, coupled with density functional theory (DFT) and ab initio computation. The hole-burning experiments indicate the population of a single conformer only, in each of the two anomers. Their experimental and calculated infrared spectra are both consistent with a conformational assignment corresponding to the computed global minimum configuration. All three OH groups are oriented towards the oxygen atom (O1) on the anomeric carbon atom to form an all trans(ttt) counter-clockwise chain of hydrogen bonds. The mono-hydrates, alpha- and beta-pXyl(H(2)O) each populate two distinct structures in the molecular beam environment, with the water molecule inserted between OH4 and OH3 or between OH3 and OH2 in alpha-pXyl(H2O), and between OH2 and O1 in either of two alternative orientations, in beta-pXyl(H2O). In all of the mono-hydrated xyloside complexes, the water molecule inserts into the weakest link of the sugar molecules' hydrogen-bonded chain of hydroxy groups, creating a single extended chain, strengthened by co-operativity. The all-trans configuration of the xylose moiety is retained and the mono-hydrate structures correspond to those calculated to lie at the lowest relative energies.
A double-quantum filtered nuclear magnetic resonance experiment is applied to half-integer quadrupolar nuclei in solids. Filtering through a double-quantum coherence excited in a dipolar-coupled two-spin system allows spatial correlations between spins to be determined. Theoretical and experimental investigations have been carried out to optimize the excitation of double-quantum coherence between the central transitions of two neighboring nuclei while minimizing double-quantum excitation on each individual spin. The presence of the large quadrupolar coupling in the systems under study allows multiple-quantum excitation via the dipolar coupling between two spins even under conditions of magic-angle spinning, without the need for a recoupling sequence. Two-dimensional double-quantum-filtered correlation spectra have been recorded on Na23 (I=32) for Na2ZrO3 which contains three distinct Na23 sites.
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