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Electrostatic forces play a key role in mediating interactions between proteins. However, gaining quantitative insights into the complex effects of electrostatics on protein behavior has proved challenging, due to the wide palette of scenarios through which both cations and anions can interact with polypeptide molecules in a specific manner or can result in screening in solution. In this article, we have used a variety of biophysical methods to probe the steady-state kinetics of fibrillar protein self-assembly in a highly quantitative manner to detect how it is modulated by changes in solution ionic strength. Due to the exponential modulation of the reaction rate by electrostatic forces, this reaction represents an exquisitely sensitive probe of these effects in protein-protein interactions. Our approach, which involves a combination of experimental kinetic measurements and theoretical analysis, reveals a hierarchy of electrostatic effects that control protein aggregation. Furthermore, our results provide a highly sensitive method for the estimation of the magnitude of binding of a variety of ions to protein molecules.

The acylphosphatase from Sulfolobus solfataricus (Sso AcP) is a globular protein able to aggregate in vitro from a native-like conformational ensemble without the need for a transition across the major unfolding energy barrier. This process leads to the formation of assemblies in which the protein retains its native-like structure, which subsequently convert into amyloid-like aggregates. Here, we investigate the mechanism by which Sso AcP aggregates in vivo to form bacterial inclusion bodies after expression in E. coli . Shortly after the initiation of expression, Sso AcP is incorporated into inclusion bodies as a native-like protein, still exhibiting small but significant enzymatic activity. Additional experiments revealed that this overall process of aggregation is enhanced by the presence of the unfolded N-terminal region of the sequence and by destabilization of the globular segment of the protein. At later times, the Sso AcP molecules in the inclusion bodies lose their native-like properties and convert into β -sheet-rich amyloid-like structures, as indicated by their ability to bind thioflavin T and Congo red. These results show that the aggregation behavior of this protein is similar in vivo to that observed in vitro, and that, at least for a predominant part of the protein population, the transition from a native to an amyloid-like structure occurs within the aggregate state.

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 concerted action of a large number of individual molecular level events in the formation and growth of fibrillar protein structures creates a significant challenge for differentiating between the relative contributions of different self-assembly steps to the overall kinetics of this process. The characterization of the individual steps is, however, an important requirement for achieving a quantitative understanding of this general phenomenon which underlies many crucial functional and pathological pathways in living systems. In this study, we have applied a kinetic modeling approach to interpret experimental data obtained for the aggregation of a selection of site-directed mutants of the protein S6 from Thermus thermophilus. By studying a range of concentrations of both the seed structures, used to initiate the reaction, and of the soluble monomer, which is consumed during the growth reaction, we are able to separate unambiguously secondary pathways from primary nucleation and fibril elongation. In particular, our results show that the characteristic autocatalytic nature of the growth process originates from secondary processes rather than primary nucleation events, and enables us to derive a scaling law which relates the initial seed concentration to the onset of the growth phase.

The molecular chaperone αB-crystallin is a small heat-shock protein that is upregulated in response to a multitude of stress stimuli, and is found colocalized with Aβ amyloid fibrils in the extracellular plaques that are characteristic of Alzheimer's disease. We investigated whether this archetypical small heat-shock protein has the ability to interact with Aβ fibrils in vitro. We find that αB-crystallin binds to wild-type Aβ42 fibrils with micromolar affinity, and also binds to fibrils formed from the E22G Arctic mutation of Aβ42. Immunoelectron microscopy confirms that binding occurs along the entire length and ends of the fibrils. Investigations into the effect of αB-crystallin on the seeded growth of Aβ fibrils, both in solution and on the surface of a quartz crystal microbalance biosensor, reveal that the binding of αB-crystallin to seed fibrils strongly inhibits their elongation. Because the lag phase in sigmoidal fibril assembly kinetics is dominated by elongation and fragmentation rates, the chaperone mechanism identified here represents a highly effective means to inhibit fibril proliferation. Together with previous observations of αB-crystallin interaction with α-synuclein and insulin fibrils, the results suggest that this mechanism is a generic means of providing molecular chaperone protection against amyloid fibril formation.

Protein misfolding and aggregation are interconnected processes involved in a wide variety of nonneuropathic, systemic, and neurodegenerative diseases. More generally, if mutations in sequence or changes in environmental conditions lead to partial unfolding of the native state of a protein, it will often aggregate, sometimes into well-defined fibrillar structures. A great deal of interest has been directed at discovering the characteristic features of metastable partially unfolded states that precede the aggregated states of proteins. In this work, human muscle acylphosphatase (AcP) has been first destabilized, by addition of urea or by means of elevated temperatures, and then incubated in the presence of different concentrations of 2,2,2, trifluoroethanol ranging from 5% to 25% (v/v). The results show that AcP is able to form both fibrillar and nonfibrillar aggregates with a high β-sheet content from partially unfolded states with very different structural features. Moreover, the presence of α-helical structure in such a state does not appear to be a fundamental determinant of the ability to aggregate. The lack of ready aggregation under some of the conditions examined here is attributable primarily to the intrinsic properties of the solutions rather than to specific structural features of the partially unfolded states that precede aggregation. Aggregation appears to be favored when the solution conditions promote stable intermolecular interactions, particularly hydrogen bonds. In addition, the structures of the resulting aggregates are largely independent of the conformational properties of their soluble precursors.

Recent advances in experimental and computational methods have made it possible to determine with considerable accuracy the structures whose formation is rate limiting for the folding of some small proteins—the transition state ensemble, or TSE. We present a method to analyze and validate all-atom models of such structures. The method is based on the comparison of experimental data with the computation of the change in free energy of the TSE resulting from specific mutations. Each mutation is modeled individually in all members of an ensemble of transition state structures using a method originally developed to predict mutational changes in the stability of native proteins. We first apply this method to six proteins for which we have determined the TSEs with a technique that uses experimental mutational data (Φ-values) as restraints in the structure determination and find a highly significant correlation between the calculated free energy changes and those derived from experimental kinetic data. We then use the procedure to analyze transition state structures determined by molecular dynamics simulations of unfolding, again finding a high correlation. Finally, we use the method to estimate changes in folding rates of several hydrophobic core mutants of Fyn SH3. Taken together, these results show that the procedure developed here is a tool of general validity for analyzing, assessing, and improving the quality of the structures of transition states for protein folding.

Protein conformational diseases represent a class of pathologies in which specific peptides or proteins form aberrant self-assemblies that constitute the hallmark of several neurodegenerative diseases. Specifically, the formation of intra-neuronal inclusions of the protein α-synuclein (αSyn) is associated with the pathogenesis of Parkinson's disease (PD).A great interest is in the early stages of αSyn aggregation, for which soluble monomeric proteins are converted into fibrillar nanostructures. It has been shown that at these stages many parallel and competing pathways take contemporaneously place and it is currently very difficult to address on these mechanisms by using standard techniques of molecular investigations. In order to overcome the limitations of standard approaches, we employed ensemble-averaged kinetic studies coupled with microdloplet technology in order to characterize the primary nucleation early stages of αSyn amyloid formation and therefore to elucidate the fundamental mechanisms underlying this phenomenon. Testing different aggregation conditions, we have been able to understand that the primary nucleation mechanism underlying αSyn aggregation is not homogeneous, whereas it is catalysed by different factors, including air/water surface interactions.The full characterization of all the processes involved in the aggregation mechanism of αSyn will be fundamental for devising new and innovative therapeutic strategies against PD. Indeed, based on our analysis, we expect that it will be possible to design and screen pharmacological compounds able to selectively inhibit the nucleation steps that trigger either the overall of the process or specifically the formation of the toxic aggregated species.

Understanding and control the aggregation of biomolecules at the molecular level can open attractive possibilities to correct dysfunctional cell behaviour. For instance, the inhibition of protein aggregation is emerging as a potential attractive therapeutic strategy against several neurodegenerative disorders. For the development of successful treatments, it is crucial to achieve a controlled intervention on specific toxic species. In this perspective, an understanding of the molecular inhibition mechanism of protein self-assembly is of fundamental importance but remains challenging to achieve.In this work, we demonstrate how chemical kinetic analysis can be applied to elucidate the molecular mechanism of inhibition of several classes of compounds such as small chemical molecules, nanoparticles, peptides and proteins. By applying a population balance model we show how it is possible to obtain information on the specific inhibited microscopic event and on the specific protein target species responsible for this inhibition. We demonstrate the potentiality of the approach by analyzing the inhibition mechanism of selected chaperones, protein regulators of the proteostais network and relevant naturally occurring inhibitors of protein aggregation, on the aggregation of a yeast prion protein and of Abeta42, the peptide involved in Alzheimer's disease. In addition, we discuss relevant implications of the controlled inhibition of protein aggregation in the engineering of the fibrillation reaction pathway and in the development of effective therapeutic strategies.

An experimental procedure has been devised to record simultaneously fluorescence intensity and fluorescence anisotropy. A photoelastic modulator on the excitation beam enables the anisotropy signal to be recorded in one pass using a single photomultiplier tube and eliminates the need for a polarizer on the emission path. In conjunction with a stopped-flow mixer, providing a time-resolved capability, this procedure was used to study the refolding of apo α-lactalbumin following dilution from guanidinium chloride. Although the fluorescence intensity does not change detectably, the fluorescence anisotropy was found to resolve the conformational changes occurring between the initial unfolded state and the molten globule state formed either kinetically during refolding at pH 7.0 or at equilibrium at pH 2.0 (A-state). This result provides further evidence that fluorescence anisotropy is a valuable probe of protein structural transitions and that the information it provides concerning the rotational mobility of a fluorophore can be complementary to the information about the local environment provided by fluorescence intensity.

The self-assembly and aggregation of insulin molecules has been investigated by means of nanoflow electrospray mass spectrometry. Hexamers of insulin containing predominantly two, but up to four, Zn2+ ions were observed in the gas phase when solutions at pH 4.0 were examined. At pH 3.3, in the absence of Zn2+, dimers and tetramers are observed. Spectra obtained from solutions of insulin at millimolar concentrations at pH 2.0, conditions under which insulin is known to aggregate in solution, showed signals from a range of higher oligomers. Clusters containing up to 12 molecules could be detected in the gas phase. Hydrogen exchange measurements show that in solution these higher oligomers are in rapid equilibrium with monomeric insulin. At elevated temperatures, under conditions where insulin rapidly forms amyloid fibrils, the concentration of soluble higher oligomers was found to decrease with time yielding insoluble high molecular weight aggregates and then fibrils. The fibrils formed were examined by electron microscopy and the results show that the amorphous aggregates formed initially are converted to twisted, unbranched fibrils containing several protofilaments. Fourier transform infrared spectroscopy shows that both the soluble form of insulin and the initial aggregates are predominantly helical, but that formation of β-sheet structure occurs simultaneously with the appearance of well-defined fibrils.

Human cystatin C (HCC), an inhibitor of cysteine proteases, is also involved in amyloidogenic processes within the human body. In the crystal structure, as in solution, wild-type (WT) cystatin C dimerizes via a domain swapping mechanism [1,2]. The same process has also been shown to be important for protein oligomerization [2]. Aggregation of HCC results in a heterogeneous mixture of species, both oligomeric and fibrillar, with varying shapes, sizes and molecular weights.The goal of our study was to characterize the assembly process of potentially neurotoxic oligomers of WT and variant HCC in solution. The oligomerization processes of several point mutation variants of HCC (V57N, V57P, V57D, V57G and L68V), with specific dimerization propensities [3], were compared to the WT protein. Prepared oligomers were purified by size-exclusion chromatography and using TEM and AFM studies, we observed characteristic donut-like structures along with fibrils and we determined the size distribution of cystatin C oligomers obtained from the WT and variant forms. Independently, the oligomerization process was analyzed by fluorescence spectroscopy and visualized by native electrophoresis. Additionally, induced secondary structure changes were characterised by circular dichroism (CD) and infrared (FTIR) spectroscopies. Finally, the structural studies were supplemented with the modelling of the larger HCC oligomers using molecular dynamics simulations, allowing the identification of the most stable oligomeric forms of HCC to be determined.This research project was financed by the National Science Centre (Poland), decision no. 2012/06/M/ST4/00036.[1] Janowski R, Kozak M, Jankowska E et al. (2001). Nature Struct. Mol. Biol. 8, 316.[2] Wahlbom M, Wang X, Lindstrom V, et al. (2007). J. Biol. Chem. 282, 18318.[3] Szymanska A, Jankowska E, Orlikowska M, et al. (2012) Front. Mol. Neurosci., 5, 82

The conversion of α-synuclein (αs) into oligomeric and fibrillar species and its deposition into Lewy bodies is the pathological hallmark of Parkinson's disease (PD). It is therefore of great importance to understand the mechanism of αs aggregation and its relationship to PD pathogenesis. We use single molecule fluorescence techniques with fast flow microfluidics to follow the early stages of this process in vitro. The methodology is based on the detection of fluorescent bursts from red or blue fluorophore-tagged αs species as they flow through a blue confocal laser volume. Forster Resonance Energy Transfer (FRET) occurs between blue and red fluorescently-tagged αs within the oligomers, giving rise to a signal in both the acceptor and donor channels. As only oligomers generate a coincident signal, they can be isolated from a solution which is >99% monomer, allowing their size and structure to be determined. By taking regular time-points, the kinetics of the aggregation can be deduced.This technique has allowed us to gain a unique insight into the effects of nanobodies and molecular chaperones on the aggregation of both the wild-type and pathological mutants of αs.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Amyloid fibrils are filamentous aggregates of peptides and proteins implicated in a range of neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. It has been known almost since their discovery that these β-sheet-rich proteinacious assemblies bind a range of specific dyes that, combined with other biophysical techniques, are convenient probes of the process of amyloid fibril formation. Two prominent examples of such dyes are Congo red (CR) and Thioflavin T (ThT). It has been reported that in addition to having a diagnostic role, CR is an inhibitor of the formation of amyloid structures, and these two properties have both been explained in terms of the same specific noncovalent interactions between the fibrils and the dye molecules. In this article, we show by means of quartz-crystal microbalance measurements that the binding of both ThT and CR to amyloid fibrils formed by the peptide whose aggregation is associated with Alzheimer's disease, Aβ(1–42), can be directly observed, and that the presence of CR interferes with the binding of ThT. Light scattering and fluorescence measurements confirm that an interaction exists between these dyes that can interfere with their ability to reflect accurately the quantity of amyloid material present in a given sample. Furthermore, we show that CR does not inhibit the process of amyloid fibril elongation, and therefore demonstrate the ability of the quartz-crystal microbalance method not only to detect and study the binding of small molecules to amyloid fibrils, but also to elucidate the mode of action of potential inhibitors.

The extent to which proteins aggregate into distinct structures ranging from prefibrillar oligomers to amyloid fibrils is key to the pathogenesis of many age-related degenerative diseases. We describe here for the Alzheimer's disease-related amyloid beta peptide (Abeta) an investigation of the sequence-based determinants of the balance between the formation of prefibrillar aggregates and amyloid fibrils. We show that by introducing single-point mutations, it is possible to convert the normally harmless Abeta40 peptide into a pathogenic species by increasing its relative propensity to form prefibrillar but not fibrillar aggregates, and, conversely, to abolish the pathogenicity of the highly neurotoxic E22G Abeta42 peptide by reducing its relative propensity to form prefibrillar species rather than mature fibrillar ones. This observation can be rationalized by the demonstration that whereas regions of the sequence of high aggregation propensity dominate the overall tendency to aggregate, regions with low intrinsic aggregation propensities exert significant control over the balance of the prefibrillar and fibrillar species formed, and therefore play a major role in determining the neurotoxicity of the Abeta peptide.

α-Synuclein has been widely accepted, since its discovery, as an intrinsically disordered protein that plays a central role in Parkinson's disease, as well as other neurodegenerative disorders associated with protein aggregation. Extensive recent data substantiate the pathogenicity of the early aggregates of α-synuclein, rather than the characteristic amyloid fibrils observed in the late stages of the aggregation process. Therefore, understanding the molecular steps and the mechanisms by which this natively unfolded protein aggregates is crucial for the purpose of identifying novel diagnostic and therapeutic strategies for the treatment of synucleinopathies. A powerful therapeutic approach is to target the initial events in the reaction process, in order to promote the solubility of the monomeric form of α-synuclein and prevent the formation of potentially harmful assemblies. Thus, in our study, we aim at understanding the structural properties of the monomer that determines its aggregation propensity, using nanobodies, the antigen-binding domains derived from camel heavy chain antibodies. These molecules are valuable probes for elucidating whether conformational changes in the monomeric protein cause the aggregation, as result of their exquisite specificity, high affinity and small size (14 KDa). Our strategy is based on the study of the interactions between α-synuclein and two specific nanobodies that bind to its C-terminus and modulate its fibrillation. The structure and dynamics of α-synuclein in its free and bound states are characterized via a combination of NMR spectroscopy and in silico tools. More specifically, chemical shifts measurements, RDCs and restrained Molecular Dynamic simulations are applied to provide a comprehensive energy sampling and description of the conformational ensemble populated by α-synuclein, and thus help gain detailed insight into the mechanism by which nanobodies modulate the aggregation process of α-synuclein.

The binding of sodium ions to the transmembrane channel peptide gramicidin A has permitted the use of electrospray ionization mass spectrometry to study its conformation in different solvent environments. The mass spectra of the peptide in the various solvents suggest that different conformations of gramicidin A differ in their ability to bind metal ions. The data are consistent with monomeric behavior of gramicidin A in trifluoroethanol and dimethyl sulfoxide solutions, but reveal the presence of noncovalent intermolecular interactions in ethanol solution through the observation of heterodimers formed between the naturally occurring variants of the peptide. The addition of 50% v/v of water to the ethanolic solution causes changes in the circular dichroism spectrum of the peptide, suggestive of a shift in the equilibrium mixture of conformers present toward monomeric species, a result supported by its mass spectrum. The structure of gramicidin A in trifluoroethanol has also been investigated by hydrogen exchange measurements monitored by mass spectrometry. The observation of significant protection against exchange suggests that the monomeric peptide is highly structured in trifluoroethanol. The results indicate that mass spectrometry has the potential to probe the conformational behavior of neutral hydrophobic peptides in environments that mimic their functional states.

Increasing evidence shows a strong link between the self-assembly of the amyloid beta peptide (Aβ) and the pathogenesis of Alzheimer's disease. Soluble oligomeric Aβ assemblies are thought to be the toxic species causing synaptic and neuronal injury in the patient's brain. Many inhibitors for the oligomerization and/or fibrillation process of neurodegenerative diseases have been reported, yet only little is known about the mechanistic details of these compounds. The present studies concern the interaction of one such inhibitor, lacmoid. We investigated this interaction by a broad biophysical approach revealing similar binding characteristics to Aβ as has been reported for detergents. Furthermore, we show that lacmoid has the ability to inhibit both oligomeric assembly and fibrillation of Aβ. Nuclear magnetic resonance experiments show an overall signal decrease upon addition of lacmoid while the chemical shifts only display small changes. High lacmoid concentration causes a loss of the major part of NMR signals including 1H-15N-HSQC, 1H-15N-TROSY and 1H-13C-HSQC cross-peaks. Circular Dichroism spectroscopy was applied to monitor the kinetic aggregation process of Aβ in presence of lacmoid. Low lacmoid concentrations slow down the conversion from a random coil-like towards a beta-sheet state while at high concentration lacmoid completely prohibits secondary structure changes.Taken together, these findings provide the basis for a simple model which could explain how non-specific interactions with small molecules interfere with amyloid formation. Understanding the mechanistic details is potentially helpful for future drug design of small molecule therapeutics targeting amyloid disorders, such as Alzheimer's disease.

It has been established that nascent proteins can fold concomitant with their synthesis on the ribosome and that chaperones such as Trigger Factor (TF) can interact with the growing nascent chain. The impact of the ribosome and TF on protein folding properties is still largely unknown, although recent studies suggest that in the near future high resolution information on contranslational folding will be available. Motivated by this, and with the goal of outlining plausible folding scenarios in vivo, we use coarse-grained simulations to examine folding in the presence and absence of the ribosome and TF. Using physically plausible interaction strengths between TF and the ribosome, parameterized based on experimental binding free energies, we have used replica exchange simulations to explore the impact of these in vivo actors on nascent chain folding. We find the ribosome significantly perturbs most protein folding properties, altering the distribution of folding pathways and favoring N-terminal folding of the nascent protein domain, suggesting that new scenarios of folding can occur on the ribosome. We then explore how TF alters cotranslational folding properties and discuss these findings in the context of recent NMR results.View Large Image | View Hi-Res Image | Download PowerPoint Slide

The devastating symptoms of Alzheimer's Disease (AD) have been attributed to the behavior of aggregated Amyloid-β (Aβ) peptides. Cleaved from the extra-cellular portion of the transmembrane receptor, amyloid precursor protein (APP), Aβ has a very high propensity to aggregate into fibrils with a cross-β-sheet structure. Though fibrillar plaques have been seen as a hallmark of AD, there has recently been increasing evidence suggesting that smaller soluble Aβ oligomers are the agents of neuronal toxicity. Molecular level characterization of these oligomers using conventional biochemical techniques has been very difficult as they are both short-lived and heterogeneous.In this work we have used a single molecule fluorescence microscopy technique to follow the formation and resolve distributions of these oligomers. Two-color coincidence detection (TCCD) has the ability to detect such transient complexes even when they comprise only 0.1% of the population (1, 2). In this work, we have detected and characterized the species ranging from dimers to 50-mers formed during the oligomerization of monomeric Aβ1-40 (the 40-amino acid portion of Aβ) as well as those resulting from breakage of Aβ1-40 fibrils. Through experiments tracking the formation of oligomers throughout the Aβ aggregation process in real-time, we have gained novel insights into the mechanisms of oligomer formation and fibril breakage.1. A. Orte, R. Clarke, S. Balasubramanian, D. Klenerman, Anal.Chem.78, 7707 (2006).2. A. Orte et al., Proc. Natl. Acad. Sci. U. S. A.105, 14424 (2008).

Saturation of single resonances in the proton nuclear magnetic resonance (NMR) spectrum of a protein in solution results in changes in intensities of other resonances. These changes come about either through nuclear Overhauser effects which arise because of dipolar coupling between the protons in the molecule, or through cross-saturation effects which arise when the protein fluctuates between different well defined conformations. For lysozyme from hen egg white (mol. wt. approx. 14,500) the method reveals details of the protein structure and of individual atom fluctuations.

alphaB-Crystallin is a small heat-shock protein (sHsp) that is colocalized with alpha-synuclein (alphaSyn) in Lewy bodies-the pathological hallmarks of Parkinson's disease-and is an inhibitor of alphaSyn amyloid fibril formation in an ATP-independent manner in vitro. We have investigated the mechanism underlying the inhibitory action of sHsps, and here we establish, by means of a variety of biophysical techniques including immunogold labeling and nuclear magnetic resonance spectroscopy, that alphaB-crystallin interacts with alphaSyn, binding along the length of mature amyloid fibrils. By measurement of seeded fibril elongation kinetics, both in solution and on a surface using a quartz crystal microbalance, this binding is shown to strongly inhibit further growth of the fibrils. The binding is also demonstrated to shift the monomer-fibril equilibrium in favor of dissociation. We believe that this mechanism, by which a sHsp interacts with mature amyloid fibrils, could represent an additional and potentially generic means by which at least some chaperones protect against amyloid aggregation and limit the onset of misfolding diseases.

(31)P cross-polarization/magic angle sample spinning nuclear magnetic resonance spectra have been obtained for pyridoxal 5'-phosphate (PLP) bound to glycogen phosphorylase b (GPb) in two different crystalline forms, monoclinic and tetragonal. Analysis of the intensities of the spinning sidebands in the nuclear magnetic resonance spectra has enabled estimates of the principal values of the (31)P chemical shift tensors to be obtained. Differences between the two sets of values suggest differences in the environment of the phosphate moiety of the pyridoxal phosphate in the two crystalline forms. The tensor for the tetragonal crystalline form, T state GPb, is fully consistent with those found for dianionic phosphate groups in model compounds. The spectrum for the monoclinic crystalline form, R state GPb, although closer to that of dianionic than monoanionic model phosphate compounds, deviates significantly from that expected for a simple dianion or monoanion. This is likely to result from specific interactions between the PLP phosphate group and residues in its binding site in the protein. A possible explanation for the spectrum of the monoclinic crystals is that the shift tensor is averaged by a proton exchange process between different ionization states of the PLP associated with the presence of a sulfate ion bound in the vicinity of the PLP.

The functions of proteins depend on the dynamical behavior of their native states on a wide range of timescales. To investigate these dynamics in the case of the small protein Gbeta1, we analyzed molecular dynamics simulations with the model-free approach of nuclear magnetic relaxation. We found amplitudes of fast timescale motions (sub-tau(c), where tau(c) is the rotational correlation time) consistent with S(2) obtained from spin relaxation measurements as well as amplitudes of slow timescale motions (supra-tau(c)) in quantitative agreement with S(2) order parameters derived from residual dipolar coupling measurements. The slow timescale motions are associated with the large variations of the (3)J couplings that follow transitions between different conformational substates. These results provide further characterization of the large structural fluctuations in the native states of proteins that occur on timescales longer than the rotational correlation time.

Folded proteins can access aggregation-prone states without the need for transitions that cross the energy barriers for unfolding. In this study we characterized the initial steps of aggregation from a native-like state of the acylphosphatase from Sulfolobus solfataricus (Sso AcP). Using computer simulations restrained by experimental hydrogen/deuterium (H/D) exchange data, we provide direct evidence that under aggregation-promoting conditions Sso AcP populates a conformational ensemble in which native-like structure is retained throughout the sequence in the absence of local unfolding (N), although the protein exhibits an increase in hydrodynamic radius and dynamics. This transition leads an edge strand to experience an increased affinity for a specific unfolded segment of the protein. Direct measurements by means of H/D exchange rates, isothermal titration calorimetry, and intermolecular relaxation enhancements show that after formation of N, an intermolecular interaction with an antiparallel arrangement is established between the edge strand and the unfolded segment of the protein. However, under conditions that favor the fully native state of Sso AcP, such an interaction is not established. Thus, these results reveal a novel (to our knowledge) self-assembly mechanism for a folded protein that is based on the increased flexibility of highly aggregation-prone segments in the absence of local unfolding. © 2012 Biophysical Society.

α-synuclein (α-syn) is the central protein in the etiology of Parkinson's diseases. The development of this disorder has indeed been associated with the formation of insoluble α-syn amyloid fibrils. When isolated in solution, α-syn behaves an intrinsically disordered protein, however, in vivo monomeric α-syn exists in equilibrium between free and membrane-bound states, the latter featuring an increased level of alpha-helix structure. α-syn binding to lipid membranes is a key process with functional relevance in synaptic regulation. This interaction also impacts α-syn fibril formation, i.e. from the inhibition to acceleration. Understanding the molecular basis of α-syn/membrane interaction is therefore a top current challenge. We here adopt advanced techniques of nuclear magnetic resonance to dissect the underlying principles governing this binding and its regulation under physiological conditions.

Maintaining protein solubility is fundamental to proteostasis, as the formation of diverse aggregated species is associated with a variety of cytotoxic events and disorders, including Alzheimer's and Parkinson's diseases. Increasing evidence indicates that protein aggregation can be widespread in living systems, as many different proteins aggregate upon cellular stress. The origins of this proteomic metastability, however, remain unclear, as do the reasons only certain proteins aggregate in vivo. We have applied simple models of cotranslational folding and protein supersaturation to quantify metastability at a proteome scale. We find that many proteins can shift from cotranslational to posttranslational folding because of translation kinetics, a source of metastability for nascent chains. Further, we show that the proteins most vulnerable to aggregation are those whose cellular concentrations are high relative to their intrinsic solubilities. These supersaturated proteins constitute a metastable sub-proteome involved in forming pathological assemblies in stress and ageing. We find that such proteins are overrepresented in the biochemical processes associated with neurodegenerative disorders, helping to rationalise their specific cellular pathologies. We anticipate that this type of analysis can provide a generally applicable basis for tracking the instability of proteomes in ageing, stress, and disease.View Large Image | View Hi-Res Image | Download PowerPoint Slide

The conversion of soluble proteins into amyloid fibrils and the structural characterization of these aggregates are outstanding problems in molecular biophysics. The amyloid-forming properties of the SH3 domain of the p85a subunit of phosphatidyl-inositol-3-kinase (PI3-SH3), an 86-residue protein, have been extensively studied in vitro by molecular biology methods. However the structure of the protein in fibril form remains unknown, and the specific structural interactions that drive its quaternary arrangement into fibrils have not been elucidated in detail. Since fibrils are insoluble and yield limited x-ray diffraction data, solid-sate NMR spectroscopy is the best-suited method with which to obtain high-resolution structural information of these systems.We present the results of solid-state NMR experiments aimed at elucidating the structure of PI3-SH3 in amyloid fibril form. The spectra of uniformly labeled PI3-SH3 present generally narrow 13C and 15N linewidths, which is characteristic of a high degree of microscopic order and structural homogeneity. Furthermore, samples grown using [2-13C] glycerol as the sole carbon source simplify the spectra and facilitate the observation of long-range internuclear correlations. Employing these alternating labeled samples and a simple data collection protocol consisting of two-dimensional homonuclear and heteronuclear correlation experiments, we have assigned ∼75% of the resonances, with the majority of unassigned residues being absent from dipolar correlation spectra due to dynamic or static disorder. Chemical shift analysis is then used to predict secondary structure elements in the PI3-SH3 sequence. The structural characteristics of PI3-SH3 fibrils revealed by solid-state NMR are described in relation to known structural and mechanistic data from previous PI3-SH3 studies.

Aggregation and deposition of TAR DNA-binding protein 43 (TDP-43) in motor neurons is a key pathological feature in nearly all amyotrophic lateral sclerosis (ALS) cases. However, little is known about the mechanism of TDP-43 aggregate formation, in particular how the protein structure changes over time and how this correlates with changes in cellular localisation. To date, a detailed analysis of the dynamic processes leading to TDP-43 aggregate deposits in live cells has not been reported. We have established neuronal cell lines that express the full length or C-terminal fragment (residue274-414) of TDP-43 tagged with a tetracysteine (TC) motif that binds specifically to biarsenical dyes (FlAsH, ReAsH), and we are comparing this to a cell model expressing TDP-43 fused to green fluorescent protein (GFP). Unlike GFP which is 27 kDa in size and thus, much larger than the C-terminal fragment, the TC-tag and biarsenical dyes are relatively small (∼1.1kDa); therefore, are less likely to interfere with the distribution and function of recombinant proteins. Using confocal microscopy and intermolecular FRET analysis, we demonstrate that biarsenical labelling technique enable the visualisation of TDP-43 localisation for the monomers and aggregate formed within live cells; in particular we can monitor the changes in distribution and aggregation as a function of time. Furthermore, using this approach we are able to compare, in a quantitative manner, the behaviour of ALS-associated variants of TDP-43 (A315T, M337V) that have been reported to possess enhanced aggregation as compared to the wild-type protein. Such models will prove useful towards investigating how changes in the structure of TDP-43 influences its localisation and aggregation under both physiological and cell stress conditions, and how these processes may relate to the pathogenicity and progression of ALS.

The pathological hallmark of Parkinson's disease is the presence of insoluble protein deposits in the brain, which are formed when specific protein molecules misfold and aggregate into highly ordered fibrils. In Parkinson's disease, the deposits are primarily made up of alpha-synuclein, a protein whose major function is not fully known. Rather than the fibrils themselves being toxic, evidence now points towards the smaller, soluble oligomers formed in the initial stages of the process as being the culprit. We have developed a novel single-molecule fluorescence technique to detect and characterise the oligomers of alpha-synuclein. Using this methodology, we are able to identify the cytotoxic species, and apply these species to primary neuronal cultures to investigate their damaging effects.