Marco Brucale

Conformational Equilibria in Monomeric α-Synuclein at the Single-Molecule Level

Human α-Synuclein (αSyn) is a natively unfolded protein whose aggregation into amyloid fibrils is involved in the pathology of Parkinson disease. A full comprehension of the structure and dynamics of early intermediates leading to the aggregated states is an unsolved problem of essential importance to researchers attempting to decipher the molecular mechanisms of αSyn aggregation and formation of fibrils. Traditional bulk techniques used so far to solve this problem point to a direct correlation between αSyns unique conformational properties and its propensity to aggregate, but these techniques can only provide ensemble-averaged information for monomers and oligomers alike. They therefore cannot characterize the full complexity of the conformational equilibria that trigger the aggregation process. We applied atomic force microscopy–based single-molecule mechanical unfolding methodology to study the conformational equilibrium of human wild-type and mutant αSyn. The conformational heterogeneity of monomeric αSyn was characterized at the single-molecule level. Three main classes of conformations, including disordered and “β-like” structures, were directly observed and quantified without any interference from oligomeric soluble forms. The relative abundance of the “β-like” structures significantly increased in different conditions promoting the aggregation of αSyn: the presence of Cu2+, the pathogenic A30P mutation, and high ionic strength. This methodology can explore the full conformational space of a protein at the single-molecule level, detecting even poorly populated conformers and measuring their distribution in a variety of biologically important conditions. To the best of our knowledge, we present for the first time evidence of a conformational equilibrium that controls the population of a specific class of monomeric αSyn conformers, positively correlated with conditions known to promote the formation of aggregates. A new tool is thus made available to test directly the influence of mutations and pharmacological strategies on the conformational equilibrium of monomeric αSyn.

Single-Molecule Studies of Intrinsically Disordered Proteins

Marco Brucale,*,† Benjamin Schuler,*,‡ and Bruno Samorì* †Institute for the Study of Nanostructured Materials (ISMN), Italian National Council of Research (CNR), Area della Ricerca Roma1, Via Salaria km 29.3 00015 Monterotondo (Rome), Italy ‡Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland Department of Pharmacy and Biotechnology, University of Bologna, Via S. Giacomo 11, 40126 Bologna, Italy

Pathogenic Mutations Shift the Equilibria of α‐Synuclein Single Molecules towards Structured Conformers

α‐synuclein (α‐Syn) is an abundant brain protein whose mutations have been linked to early‐onset Parkinsons disease (PD). We recently demonstrated, by means of a single‐molecule force spectroscopy (SMFS) methodology, that the conformational equilibrium of monomeric wild‐type (WT) α‐Syn shifts toward β‐containing structures in several unrelated conditions linked to PD pathogenicity. Herein, we follow the same methodology previously employed for WT α‐Syn to characterize the conformational heterogeneity of pathological α‐Syn mutants A30P, A53T, and E46K. Contrary to the bulk ensemble‐averaged spectroscopies so far employed to this end by different authors, our single‐molecule methodology monitored marked differences in the conformational behaviors of the mutants with respect to the WT sequence. We found that all the mutants have a much higher propensity than the WT to adopt a monomeric compact conformation that is compatible with the acquiring of β structure. Mutants A30P and A53T show a similar conformational equilibrium that is significantly different from that of E46K. Another class of conformations, stabilized by mechanically weak interactions (MWI), shows a higher variety in the mutants than in the WT protein. In the A30P mutant these interactions are relatively stronger, and therefore the corresponding conformations are possibly more structured. The more structured and globular conformations of the mutants can explain their higher propensity to aggregate with respect to the WT.

The dynamic properties of an intramolecular transition from DNA duplex to cytosine–thymine motif triplex

We here report that the formation and breakdown of an intramolecular cytosine-thymine (CT) motif DNA triple-helix can be performed repeatedly, quickly and independently of its local concentration without performance reduction over successive cycles; as a consequence, we propose that this set of characteristics makes the DNA duplex-triplex transition an ideal candidate to power simple nanometer-scale devices capable of maintaining effective performance regardless of their local concentration.

The chaperone–like protein 14-3-3η interacts with human α-synuclein aggregation intermediates rerouting the amyloidogenic pathway and reducing α-synuclein cellular toxicity

Familial and idiopathic Parkinsons disease (PD) is associated with the abnormal neuronal accumulation of α-synuclein (aS) leading to β-sheet-rich aggregates called Lewy Bodies (LBs). Moreover, single point mutation in aS gene and gene multiplication lead to autosomal dominant forms of PD. A connection between PD and the 14-3-3 chaperone-like proteins was recently proposed, based on the fact that some of the 14-3-3 isoforms can interact with genetic PD-associated proteins such as parkin, LRRK2 and aS and were found as components of LBs in human PD. In particular, a direct interaction between 14-3-3η and aS was reported when probed by co-immunoprecipitation from cell models, from parkinsonian brains and by surface plasmon resonance in vitro. However, the mechanisms through which 14-3-3η and aS interact in PD brains remain unclear. Herein, we show that while 14-3-3η is unable to bind monomeric aS, it interacts with aS oligomers which occur during the early stages of aS aggregation. This interaction diverts the aggregation process even when 14-3-3η is present in sub-stoichiometric amounts relative to aS. When aS level is overwhelmingly higher than that of 14-3-3η, the fibrillation process becomes a sequestration mechanism for 14-3-3η, undermining all processes governed by this protein. Using a panel of complementary techniques, we single out the stage of aggregation at which the aS/14-3-3η interaction occurs, characterize the products of the resulting processes, and show how the processes elucidated in vitro are relevant in cell models. Our findings constitute a first step in elucidating the molecular mechanism of aS/14-3-3η interaction and in understanding the critical aggregation step at which 14-3-3η has the potential to rescue aS-induced cellular toxicity.

Extracellular production of tellurium nanoparticles by the photosynthetic bacterium Rhodobacter capsulatus.

The toxic oxyanion tellurite (TeO3(2-)) is acquired by cells of Rhodobacter capsulatus grown anaerobically in the light, via acetate permease ActP2 and then reduced to Te(0) in the cytoplasm as needle-like black precipitates. Interestingly, photosynthetic cultures of R. capsulatus can also generate Te(0) nanoprecipitates (TeNPs) outside the cells upon addition of the redox mediator lawsone (2-hydroxy-1,4-naphtoquinone). TeNPs generation kinetics were monitored to define the optimal conditions to produce TeNPs as a function of various carbon sources and lawsone concentration. We report that growing cultures over a 10 days period with daily additions of 1mM tellurite led to the accumulation in the growth medium of TeNPs with dimensions from 200 up to 600-700 nm in length as determined by atomic force microscopy (AFM). This result suggests that nucleation of TeNPs takes place over the entire cell growth period although the addition of new tellurium Te(0) to pre-formed TeNPs is the main strategy used by R. capsulatus to generate TeNPs outside the cells. Finally, X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FT-IR) analysis of TeNPs indicate they are coated with an organic material which keeps the particles in solution in aqueous solvents.

Covalent α-synuclein dimers: chemico-physical and aggregation properties.

The aggregation of α-synuclein into amyloid fibrils constitutes a key step in the onset of Parkinsons disease. Amyloid fibrils of α-synuclein are the major component of Lewy bodies, histological hallmarks of the disease. Little is known about the mechanism of aggregation of α-synuclein. During this process, α-synuclein forms transient intermediates that are considered to be toxic species. The dimerization of α-synuclein could represent a rate-limiting step in the aggregation of the protein. Here, we analyzed four covalent dimers of α-synuclein, obtained by covalent link of the N-terms, C-terms, tandem cloning of two sequences and tandem juxtaposition in one protein of the 1–104 and 29–140 sequences. Their biophysical properties in solution were determined by CD, FT-IR and NMR spectroscopies. SDS-induced folding was also studied. The fibrils formation was analyzed by ThT and polarization fluorescence assays. Their morphology was investigated by TEM and AFM-based quantitative morphometric analysis. All dimers were found to be devoid of ordered secondary structure under physiological conditions and undergo α-helical transition upon interaction with SDS. All protein species are able to form amyloid-like fibrils. The reciprocal orientation of the α-synuclein monomers in the dimeric constructs affects the kinetics of the aggregation process and a scale of relative amyloidogenic propensity was determined. Structural investigations by FT IR spectroscopy, and proteolytic mapping of the fibril core did not evidence remarkable difference among the species, whereas morphological analyses showed that fibrils formed by dimers display a lower and diversified level of organization in comparison with α-synuclein fibrils. This study demonstrates that although α-synuclein dimerization does not imply the acquisition of a preferred conformation by the participating monomers, it can strongly affect the aggregation properties of the molecules. The results presented highlight a substantial role of the relative orientation of the individual monomer in the definition of the fibril higher structural levels.

Single‐Molecule‐Level Evidence for the Osmophobic Effect

Organic osmolytes are low-molecular-weight osmotically active compounds, which are ubiquitous in living systems and are able to modulate protein stability. Among them, those that act as folding agonists, enhancing the stability of the native structure of proteins, such as trimethylamine N-oxide, betaine, sarcosine, proline, trehalose, sucrose, glycerol, sorbitol, and dimethylsulphoxide (DMSO), are collectively called protecting osmolytes or “chemical chaperones”. One rather puzzling feature of these compounds is that they are able to affect the folding of very diverse proteins in similar ways, suggesting that they might act according to a general mechanism, in contrast to the more specific mechanisms employed by chaperone proteins. In fact, the most widely accepted theory to rationalize their mode of action proposes that the addition of a protecting osmolyte to water as a cosolvent results in diminished solvent quality for the protein backbone, thus making intra-peptide backbone–backbone hydrogen bonds energetically more favorable than those between the backbone and the solvent. This effect, known as the osmophobic effect, implies that protecting osmolytes have a universal, indirect mode of action, which does not entail the presence of any specific binding sites for the osmolyte on the protein in any of its states, including the folding/unfolding transition state. On the other hand, evidence of such a direct participation have been recently provided by experimental studies for specific protein–osmolyte combinations. Herein, we provide experimental evidence, at the singlemolecule level, that the osmolyte DMSO protects the native state of a globular protein against mechanical unfolding without any active complexation of the osmolyte molecules into its unfolding transition state. Apart from slowing down the spontaneous unfolding rate of the protein, we show that the osmolyte also simultaneously accelerates its folding rate. The kinetic description of the observed stabilization mechanism strongly supports a backbone-based theory of the osmophobic effect. We employed atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) to characterize the effect of DMSO on the folding and unfolding kinetics of a globular protein domain, namely the B1 immunoglobulin binding domain of protein G from streptococcus (herein referred to as GB1), which behaves as a two-state folding protein on AFM experimental timescales. SMFS mechanical unfolding and refolding experiments were performed on polyprotein constructs made up of either eight or sixteen tandem repeats of the GB1 domain. We used buffered solutions with five different concentrations of DMSO ranging from 0% to 50 % v/v (see the Supporting Information, Section 1 for the detailed SMFS experimental methods, and Section 2 for preparation of protein constructs). The unfolding experiments were performed using three independent and complementary SMFS modes of operation (Figure 1), which consistently led to the same result: in the presence of DMSO, the spontaneous unfolding rate of GB1 at zero applied force is negatively correlated with DMSO concentration, meaning that DMSO kinetically protects the folded state against unfolding. This protection manifests itself as an increase of the average mechanical unfolding forces at all loading rates in the velocity clamp SMFS mode (Figure 2a), and a decrease of the force-dependent unfolding rates in force ramp and force clamp experiments (Supporting Information, Section 4 and Figure S1). The refolding experiments were instead performed using a variable time lapse, double pulse procedure 24] and showed that DMSO increases the spontaneous folding rate of GB1 (please refer to Supporting Information, Section 5 for the experimental details on this procedure). The data (Figure 2a) makes it possible to map the mechanical unfolding energy landscape of GB1 at each investigated DMSO concentration, extracting (as detailed elsewhere) the two fundamental kinetic parameters of the [*] D. Aioanei, Dr. A. Rampioni, Prof. B. Samor , Dr. M. Brucale Dipartimento di Biochimica, Universit di Bologna Via Irnerio 48, 40126 Bologna (Italy) and S3 Center of Nanostructures and Biosystems at Surfaces Istituto di Nanoscienze—CNR (Italy)

Observing the osmophobic effect in action at the single molecule level

Protecting osmolytes are widespread small organic molecules able to stabilize the folded state of most proteins against various denaturing stresses in vivo. The osmophobic model explains thermodynamically their action through a preferential exclusion of the osmolyte molecules from the protein surface, thus favoring the formation of intrapeptide hydrogen bonds. Few works addressed the influence of protecting osmolytes on the protein unfolding transition state and kinetics. Among those, previous single molecule force spectroscopy experiments evidenced a complexation of the protecting osmolyte molecules at the unfolding transition state of the protein, in apparent contradiction with the osmophobic nature of the protein backbone. We present single‐molecule evidence that glycerol, which is a ubiquitous protecting osmolyte, stabilizes a globular protein against mechanical unfolding without binding into its unfolding transition state structure. We show experimentally that glycerol does not change the position of the unfolding transition state as projected onto the mechanical reaction coordinate. Moreover, we compute theoretically the projection of the unfolding transition state onto two other common reaction coordinates, that is, the number of native peptide bonds and the weighted number of native contacts. To that end, we augment an analytic Ising‐like protein model with support for group‐transfer free energies. Using this model, we find again that the position of the unfolding transition state does not change in the presence of glycerol, giving further support to the conclusions based on the single‐molecule experiments. Proteins 2011;

Single molecule fluorescence spectroscopy of pH sensitive oligonucleotide switches

Several authors demonstrated that an oligonucleotide based pH-sensitive construct can act as a switch between an open and a closed state by changing the pH. To validate this process, specially designed fluorescence dye-quencher substituted oligonucleotide constructs were developed to probe the switching between these two states. This paper reports on bulk and single molecule fluorescence investigations of a duplex-triplex pH sensitive oligonucleotide switch. On the bulk level, only a partial quenching of the fluorescence is observed, similarly to what is observed for other published switches and is supposed to be due to intermolecular interactions between oligonucleotide strands. On the single molecule level, each DNA-based nanometric construct shows a complete switching. These observations suggest the tendency of the DNA construct to associate at high concentration.