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The Hsp70 family of chaperones plays an essential role in suppressing protein aggregation in the cell. Here we investigate the factors controlling the intrinsic ability of human Hsp70 to inhibit the elongation of amyloid fibrils formed by the Parkinson's disease-related protein α-synuclein. Using kinetic analysis, we show that Hsp70 binds preferentially to α-synuclein fibrils as a consequence of variations in the association and dissociation rate constants of binding to the different aggregated states of the protein. Our findings illustrate the importance of the kinetics of binding of molecular chaperones, and also of potential therapeutic molecules, in the efficient suppression of specific pathogenic events linked to neurodegeneration.

Oligomerization in the heat shock protein (Hsp) 70 family has been extensively documented both in vitro and in vivo, although the mechanism, the identity of the specific protein regions involved and the physiological relevance of this process are still unclear. We have studied the oligomeric properties of a series of human Hsp70 variants by means of nanoelectrospray ionization mass spectrometry, optical spectroscopy and quantitative size exclusion chromatography. Our results show that Hsp70 oligomerization takes place through a specific interaction between the interdomain linker of one molecule and the substrate-binding domain of a different molecule, generating dimers and higher-order oligomers. We have found that substrate binding shifts the oligomerization equilibrium towards the accumulation of functional monomeric protein, probably by sequestering the helical lid sub-domain needed to stabilize the chaperone: substrate complex. Taken together, these findings suggest a possible role of chaperone oligomerization as a mechanism for regulating the availability of the active monomeric form of the chaperone and for the control of substrate binding and release. © 2013 Aprile et al., FAA was recipient of a graduate fellowship from the Italian Ministry of Education, University and Research. AD is grateful for support from Murray Edwards College, Cambridge, through a Junior Research Fellowship. FS is a Sir Henry Wellcome Fellow. CR acknowledges financial support by the Spanish Ministry of Health according to the 'Plan Nacional de I+D+I 2008-2011', through ISCIII with cofunding by FEDER (CP10/00527). JLPB is a Royal Society University Research Fellow. FAA and PT are grateful for support from Regione Lombardia (NEDD and >Network Tecnologico integrato per lo studio proteomico e trascrittomico di malattie neurodegenerative correlate a deposizioni di amiloidi>). CMD acknowledges support from BBSRC (BB/E019927/1), the Wellcome Trust (094425/Z/10/Z), the European Commission (project LSHM-CT-2006-037525). NC acknowledges support from Human Frontiers Science Program (HFSP) through a Long-term Fellowship (LT000795/2009).

The conversion of human lysozyme into amyloid fibrils is associated with a rare but fatal hereditary form of nonneuropathic systemic amyloidosis. The accumulation of large amounts of aggregated protein is thought to be initiated by the formation of transient intermediate species of disease-related lysozyme variants, essentially due to the loss of global cooperativity under physiologically relevant conditions. Interestingly, all five naturally occurring, amyloidogenic, single-point mutations are located in the β-domain of lysozyme, the region that is predominantly unfolded during the formation of the transient intermediate species. Given the lack of known naturally occurring, amyloidogenic, single-point mutations in the α-domain, we chose three specific mutations to address the effects that location may have on native-state dynamics, as studied by hydrogen-deuterium (HD) exchange experiments analyzed by NMR spectroscopy, and mass spectrometry. We compared the effect of a destabilizing α-domain mutation (I23A) with that of the well-characterized I59T β-domain variant. We also investigated the effect of a mutation that has minor effects on native-state stability at the domain interface (I56V) and compared it with that of a variant with similar stability within the C-helix (I89V). We show that when variants have similar reduced native-state stabilities, the location of the mutation (I23A versus I59T) is crucial to the native-state dynamics, with the α-domain mutation having a significantly lower ability to populate transient intermediate species under physiologically relevant conditions. Interestingly, the mutation at the interface (I56V) has a greater effect in facilitating the formation of transient intermediate species at elevated temperatures compared with the variants containing α-domain mutations, even though this mutation results in only minor changes to the native-state stability of lysozyme. These findings reveal that the location of specific mutations is an important factor in determining the native-state dynamical properties of human lysozyme in the context of its propensity to populate the aggregation-prone transient intermediate species associated with pathogenic amyloid formation.

The partial unfolding of human lysozyme underlies its conversion from the soluble state into amyloid fibrils observed in a fatal hereditary form of systemic amyloidosis. To understand the molecular origins of the disease, it is critical to characterize the structural and physicochemical properties of the amyloidogenic states of the protein. Here we provide a high-resolution view of the unfolding process at low pH for three different lysozyme variants, the wild-type protein and the mutants I56T and I59T, which show variable stabilities and propensities to aggregate in vitro. Using a range of biophysical techniques that includes differential scanning calorimetry and nuclear magnetic resonance spectroscopy, we demonstrate that thermal unfolding under amyloidogenic solution conditions involves a cooperative loss of native tertiary structure, followed by progressive unfolding of a compact, molten globule-like denatured state ensemble as the temperature is increased. The width of the temperature window over which the denatured ensemble progressively unfolds correlates with the relative amyloidogenicity and stability of these variants, and the region of lysozyme that unfolds first maps to that which forms the core of the amyloid fibrils formed under similar conditions. Together, these results present a coherent picture at atomic resolution of the initial events underlying amyloid formation by a globular protein.

Enzymes can increase the rate of biomolecular reactions by several orders of magnitude. Although the steps of substrate capture and product release are essential in the enzymatic process, complete atomic-level descriptions of these steps are difficult to obtain because of the transient nature of the intermediate conformations, which makes them largely inaccessible to standard structure determination methods. We describe here the determination of the structure of a low-population intermediate in the product release process by human lysozyme through a combination of NMR spectroscopy and molecular dynamics simulations. We validate this structure by rationally designing two mutations, the first engineered to destabilise the intermediate and the second to stabilise it, thus slowing down or speeding up, respectively, product release. These results illustrate how product release by an enzyme can be facilitated by the presence of a metastable intermediate with transient weak interactions between the enzyme and product. DOI: http://dx.doi.org/10.7554/eLife.02777.001, eLife digest Enzymes are proteins that catalyse biochemical reactions. They bind to their target molecules—known as substrates—and help to change them to make ‘products’. Afterwards, the products are released and the enzymes are free to bind to the next molecules. To perform this activity, an enzyme can change its structure several times, but it has been challenging to characterise the intermediate shapes because of their transient nature. De Simone et al. took advantage of a technique called nuclear magnetic resonance spectroscopy to get a better look at the structures adopted by the human enzyme lysozyme. This enzyme helps to protect us from bacterial infections because it breaks the links between peptidoglycan molecules, which make up the wall that surrounds bacterial cells. The experiments show that two ‘arms’ in the lysozyme structure move to create an intermediate shape during the final step—the release of the product—in the chemical reaction. This type of flexibility gives the enzyme the ability to tightly bind the peptidoglycan at the start and to let go of the product when the chemical reaction is complete. Next, to confirm their findings, De Simone et al. examined what happened when they introduced particular mutations in the gene that makes lysozyme. The first mutation was meant to destabilise the intermediate shape of the enzyme, which resulted in the enzyme binding more tightly to the peptidoglycan in the final step and releasing it more slowly. A second mutation was made to stabilize the structure of the intermediate shape, which, as expected, allowed lysozyme to release the peptidoglycan more quickly. De Simone et al.'s findings explain how intermediate shapes can be involved in the release of the product from lysozyme and other enzymes. The next challenges will be to characterise the structure of the intermediate shape that binds to the substrate and, more generally, to extend this type of approach to other enzymes. DOI: http://dx.doi.org/10.7554/eLife.02777.002

Solubility is a key requirement for the functioning of a protein within the complex network of cellular components. A class of highly debilitating disorders, including Alzheimer s and Parkinson s diseases, is related to the loss of solubility of peptides and proteins that is accompanied by their aggregation into ordered amyloid fibrils. It has been found that, under at least some physiological conditions, these aggregates are thermodynamically more stable than the native forms of biological polypeptides. This finding raises questions as to the factors governing the crucial ability of native proteins to remain soluble even under conditions where they do not necessarily correspond to global minima on free energy landscapes. In order to address this question, we have studied in detail the kinetics of elongation of amyloid fibrils formed by a wide range of polypeptides. The formation of amyloid fibrils from soluble protein molecules involves at least a primary nucleation step, an elongation step and, in general, a secondary nucleation process such as fibril fragmentation. In addition, multiple interconverting oligomeric intermediates can be involved. Measurements of amyloid growth in bulk solution often reflect all of these processes, and it can therefore be extremely challenging to determine accurately the concentrations of the different species and the rate constants for the individual elementary steps. In order to overcome these difficulties, surface-based sensing techniques, notably those based on quartz crystal microbalance (QCM) measurements, have been developed in recent years, by which the growth of a constant, surfacebound ensemble of fibrils can be monitored. These methods make use of the fact that in the presence of preexisting fibrils, aggregation can be highly accelerated through seeding. This seeding process corresponds to the elongation of existing fibrils and can be well described as diffusional motion over a single free energy barrier, involving no intermediate species between monomeric and fibrillar peptide. The elongation of the fibrils is monitored through the increase in hydrodynamic mass bound to the quartz crystal, as the rate of change of the resonant frequency is proportional to the average elongation rate of the fibrils. The opportunity to image the sensor surface enables an estimation of the surface density of fibrils, an important factor in the determination of the rate constants in this bimolecular reaction, the overall rate of which depends on both the concentration of soluble protein and the number of available fibril ends. In addition, the lengths of the fibrils before and after an experiment can be compared and therefore an independent measurement of the length increase can be made and used to calibrate the frequency response of the microbalance. The covalent irreversible attachment of the preformed fibrils to the sensor surface and the subsequent passivation of the remaining surface, as well as the short duration of individual experiments, ensure that only fibril elongation is measured, and that primary and secondary nucleation events can be neglected; this selectivity is confirmed by the high reproducibility of the data obtained from QCM measurements. The starting point of a systematic study of the energy barriers that separate the soluble from the fibrillar states of a protein is the measurement of the temperature dependence of the fibril elongation rate; such an approach allows for the determination of the enthalpy of activation from an Arrhenius plot. The temperature dependence of amyloid growth has already been measured for a range of amyloidogenic peptides and proteins, and where possible these literature data are included in the analysis described herein. These published data have been acquired with a range of different techniques, mainly involving small-molecule labels such as Thioflavin-T. In such experiments, the exclusive study of the elementary elongation reaction is challenging and therefore the published values on energy barriers may in some cases refer to a combination of different elementary steps. We have, however, used the QCM approach, which is particularly suited for such measurements, to increase substantially the size of the available dataset by studying peptides and proteins of very diverse sequence that form amyloid fibrils under varied solution conditions. Figure 1 shows, as an example, raw QCM data for the temperature dependence of PI3K-SH3 amyloid fibril elongation, as well as AFM images of the QCM sensor surface. Similar experiments were performed for a range of other peptides and proteins, and the resulting Arrhenius plots are shown in Figure 2. No pronounced curvature is apparent, unlike that sometimes observed for protein folding; this [*] A. K. Buell, M. E. Welland Nanoscience Centre, University of Cambridge 11 JJ Thomson Avenue, West Cambridge CB3 0FF (UK) E-mail: mew10@cam.ac.uk

The identification of the factors that enable normally folded proteins to remain in their soluble and functional states is crucial for a comprehensive understanding of any biological system. We have determined a series of energy landscapes of the acylphosphatase from Drosophila melanogaster under a variety of conditions by combining NMR measurements with restrained molecular dynamics simulations. We thus analyzed the differences in the structures, dynamics, and energy surfaces of the protein in its soluble state or in situations where it aggregates through conformational states that have native-like structure, folding stability, and enzymatic activity. The study identifies the nature of the energy barriers that under normal physiological conditions prevent the protein ensemble from populating dangerous aggregation-prone states. We found that such states, although similar to the native conformation, have altered surface charge distribution, alternative topologies of the β-sheet region, and modified solvent exposure of hydrophobic surfaces and aggregation-prone regions of the sequence. The identified barriers allow the protein to undergo functional dynamics while remaining soluble and without a significant risk of misfolding and aggregation into nonfunctional and potentially toxic species.

We report here the detailed characterisation of a non-naturally occurring variant of human lysozyme, I59T, which possesses a destabilising point mutation at the interface of the alpha- and beta-domains. Although more stable in its native structure than the naturally occurring variants that give rise to a familial form of systemic amyloidosis, I59T possesses many attributes that are similar to these disease-associated species. In particular, under physiologically relevant conditions, I59T populates transiently an intermediate in which a region of the structure unfolds cooperatively; this loss of global cooperativity has been suggested to be a critical feature underlying the amyloidogenic nature of the disease-associated lysozyme variants. In the present study, we have utilised this variant to provide direct evidence for the generic nature of the conformational transition that precedes the ready formation of the fibrils responsible for lysozyme-associated amyloid disease. This non-natural variant can be expressed at higher levels than the natural amyloidogenic variants, enabling, for example, singly isotopically labelled protein to be generated much more easily for detailed structural studies by multidimensional NMR spectroscopy. Moreover, we demonstrate that the I59T variant can readily form fibrils in vitro, similar in nature to those of the amyloidogenic I56T variant, under significantly milder conditions than are needed for the wild-type protein.

Identifying the cause of the cytotoxicity of species populated during amyloid formation is crucial to understand the molecular basis of protein deposition diseases. We have examined different types of aggregates formed by lysozyme, a protein found as fibrillar deposits in patients with familial systemic amyloidosis, by infrared spectroscopy, transmission electron microscopy, and depolymerization experiments, and analyzed how they affect cell viability. We have characterized two types of human lysozyme amyloid structures formed in vitro that differ in morphology, molecular structure, stability, and size of the cross-β core. Of particular interest is that the fibrils with a smaller core generate a significant cytotoxic effect. These findings indicate that protein aggregation can give rise to species with different degree of cytotoxicity due to intrinsic differences in their physicochemical properties.

We describe the changes in structure and dynamics that occur in the second PDZ domain of human tyrosine phosphatase 1E upon binding the small peptide RA-GEF2 by an analysis of NMR data based on their use as ensemble-averaged restraints in molecular dynamics simulations. This approach reveals the presence of two interconnected networks of residues, the first exhibiting structural changes and the second dynamical changes upon binding, and it provides a detailed mapping of the regions of increased and decreased mobility upon binding. Analysis of the dynamical properties of the residues in these networks reveals that conformational changes are transmitted through pathways of coupled side-chain reorientations. These results illustrate how the strategy we described, in which NMR data are used in combination with molecular dynamics simulations, can be used to characterize in detail the complex organization of the changes in structure and dynamics that take place in proteins upon binding.

This article presents an analysis of the internal dynamics of the Ca2+-binding protein calbindin, based on the Networks of Coupled Rotators (NCRs) introduced recently. Several fundamental and practical issues raised by this approach are investigated. The roles of various parameters of the model are examined. The NCR model is shown to account for the modifications of the internal dynamics upon Ca2+ binding by calbindin. Two alternative strategies to estimate local internal effective correlation times of the protein are proposed, which offer good agreement between predictions and experiment.

In this article, a formal expression for the conformational entropy of a bond vector in a protein is derived using the networks of coupled rotators model for the description of internal dynamics. Analytical relationships between NMR order parameters and conformational entropies are derived, and the possibility to extract the latter from NMR experiments is discussed. These results are illustrated in the case of the calcium-binding protein calbindin. (C) 2008 American Institute of Physics.