Evidence of Orientation-Dependent Early States of Prion Protein Misfolded Structures from Single Molecule Force Spectroscopy.

Prion diseases are neurodegenerative disorders characterized by the presence of oligomers and amyloid fibrils. These are the result of protein aggregation processes of the cellular prion protein (PrPC) into amyloidal forms denoted as prions or PrPSc. We employed atomic force microscopy (AFM) for single molecule pulling (single molecule force spectroscopy, SMFS) experiments on the recombinant truncated murine prion protein (PrP) domain to characterize its conformations and potential initial oligomerization processes. Our AFM-SMFS results point to a complex scenario of structural heterogeneity of PrP at the monomeric and dimer level, like other amyloid proteins involved in similar pathologies. By applying this technique, we revealed that the PrP C-terminal domain unfolds in a two-state process. We used two dimeric constructs with different PrP reciprocal orientations: one construct with two sequential PrP in the N- to C-terminal orientation (N-C dimer) and a second one in the C- to C-terminal orientation (C-C dimer). The analysis revealed that the different behavior in terms of unfolding force, whereby the dimer placed C-C dimer unfolds at a higher force compared to the N-C orientation. We propose that the C-C dimer orientation may represent a building block of amyloid fibril formation.

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 Parkinson's 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 force spectroscopy of chimeric polyprotein constructs containing intrinsically disordered domains.

Here, we describe the single molecule force spectroscopy (SMFS)-based experimental protocol we have recently used to single out different classes of conformations in a chimeric multimodular protein containing an intrinsically disordered (human Alpha Synuclein) domain. Details are provided regarding cloning, expression and purification of the chimeric polyprotein constructs, optimal surface preparation, SMFS data collection and filtering. Although the specificity of the issue and the ensemble of nonstandard techniques needed to perform the described procedures render this a rather unorthodox protocol, it is relatively straightforward to adapt it to the study of other protein domains.

Worm-like Ising model for protein mechanical unfolding under the effect of osmolytes.

We show via single-molecule mechanical unfolding experiments that the osmolyte glycerol stabilizes the native state of the human cardiac I27 titin module against unfolding without shifting its unfolding transition state on the mechanical reaction coordinate. Taken together with similar findings on the immunoglobulin-binding domain of streptococcal protein G (GB1), these experimental results suggest that osmolytes act on proteins through a common mechanism that does not entail a shift of their unfolding transition state. We investigate the above common mechanism via an Ising-like model for protein mechanical unfolding that adds worm-like-chain behavior to a recent generalization of the Wako-Saitô-Muñoz-Eaton model with support for group-transfer free energies. The thermodynamics of the model are exactly solvable, while protein kinetics under mechanical tension can be simulated via Monte Carlo algorithms. Notably, our force-clamp and velocity-clamp simulations exhibit no shift in the position of the unfolding transition state of GB1 and I27 under the effect of various osmolytes. The excellent agreement between experiment and simulation strongly suggests that osmolytes do not assume a structural role at the mechanical unfolding transition state of proteins, acting instead by adjusting the solvent quality for the protein chain analyte.

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.

Open source platform for the execution and analysis of mechanical refolding experiments.

MOTIVATION: Single-molecule force spectroscopy has facilitated the experimental investigation of biomolecular force-coupled kinetics, from which the kinetics at zero force can be extrapolated via explicit theoretical models. The atomic force microscope (AFM) in particular is routinely used to study protein unfolding kinetics, but only rarely protein folding kinetics. The discrepancy arises because mechanical protein refolding studies are more technically challenging. RESULTS: We developed software that can drive and analyse mechanical refolding experiments when used with the commercial AFM setup 'Picoforce AFM', Bruker (previously Digital Instruments). We expect the software to be easily adaptable to other AFM setups. We also developed an improved method for the statistical characterization of protein folding kinetics, and implemented it into an AFM-independent software module. AVAILABILITY: Software and documentation are available at http://code.google.com/p/refolding under Apache License 2.0.

An open source/real-time atomic force microscope architecture to perform customizable force spectroscopy experiments.

We describe the realization of an atomic force microscope architecture designed to perform customizable experiments in a flexible and automatic way. Novel technological contributions are given by the software implementation platform (RTAI-LINUX), which is free and open source, and from a functional point of view, by the implementation of hard real-time control algorithms. Some other technical solutions such as a new way to estimate the optical lever constant are described as well. The adoption of this architecture provides many degrees of freedom in the device behavior and, furthermore, allows one to obtain a flexible experimental instrument at a relatively low cost. In particular, we show how such a system has been employed to obtain measures in sophisticated single-molecule force spectroscopy experiments [Fernandez and Li, Science 303, 1674 (2004)]. Experimental results on proteins already studied using the same methodologies are provided in order to show the reliability of the measure system.

Hooke: an open software platform for force spectroscopy.

SUMMARY: Hooke is an open source, extensible software intended for analysis of atomic force microscope (AFM)-based single molecule force spectroscopy (SMFS) data. We propose it as a platform on which published and new algorithms for SMFS analysis can be integrated in a standard, open fashion, as a general solution to the current lack of a standard software for SMFS data analysis. Specific features and support for file formats are coded as independent plugins. Any user can code new plugins, extending the software capabilities. Basic automated dataset filtering and semi-automatic analysis facilities are included. AVAILABILITY: Software and documentation are available at (http://code.google.com/p/hooke). Hooke is a free software under the GNU Lesser General Public License.

New insights for using self-assembly materials to improve the detection stability in label-free DNA-chip and immuno-sensors.

This paper examines reliable advancements in low-cost DNA- and immuno-chips. Capacitance detection was successfully chosen to develop label-free bio-chips. Probe immobilization was rigorously investigated in order to obtain reliable capacitance measurements. Protein probes immobilized by using usual alkanethiols or thiolated ssDNA probes directly immobilized on gold do not allow sufficient stable capacitance measurements. New alkanethiols improved with ethylene-glycol function are shown in this paper to be more suitable materials for capacitive bio-chip development. Atomic Force Microscopy, Quartz Crystal Microbalance, and Capacitance Measurements were used to demonstrate that ethylene-glycol alkanethiols allow high time stability, smaller errors in detection, and improved ideal behaviour of the sensing surfaces. Measured capacitance is in the range of 8-11 nF/mm(2) for antibody layers and close to 6 nF/mm(2) for DNA probes. It is in the range of 10-12 nF/mm(2) and of 4-6 nF/mm(2) for antigen and DNA detection, respectively. The percentage error in detection is highly improved and it is in the range of 11-37% and of 0,23-0,82% for antigen and DNA, respectively. The reproducibility is also improved and it is close to 0,44% for single spot measurements on ethylene-glycol alkanethiols. A molecular theory attributing these improvements to water molecules strongly coordinated by ethylene-glycol functional groups and to solution ions not entering into probe films is finally proposed.

Pathogenic mutations shift the equilibria of alpha-synuclein single molecules towards structured conformers.

Alpha-synuclein (alpha-Syn) is an abundant brain protein whose mutations have been linked to early-onset Parkinson's disease (PD). We recently demonstrated, by means of a single-molecule force spectroscopy (SMFS) methodology, that the conformational equilibrium of monomeric wild-type (WT) alpha-Syn shifts toward beta-containing structures in several unrelated conditions linked to PD pathogenicity. Herein, we follow the same methodology previously employed for WT alpha-Syn to characterize the conformational heterogeneity of pathological alpha-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 beta 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.

Maximum likelihood estimation of protein kinetic parameters under weak assumptions from unfolding force spectroscopy experiments.

Single molecule force spectroscopy (SMFS) is extensively used to characterize the mechanical unfolding behavior of individual protein domains under applied force by pulling chimeric polyproteins consisting of identical tandem repeats. Constant velocity unfolding SMFS data can be employed to reconstruct the protein unfolding energy landscape and kinetics. The methods applied so far require the specification of a single stretching force increase function, either theoretically derived or experimentally inferred, which must then be assumed to accurately describe the entirety of the experimental data. The very existence of a suitable optimal force model, even in the context of a single experimental data set, is still questioned. Herein, we propose a maximum likelihood (ML) framework for the estimation of protein kinetic parameters which can accommodate all the established theoretical force increase models. Our framework does not presuppose the existence of a single force characteristic function. Rather, it can be used with a heterogeneous set of functions, each describing the protein behavior in the stretching time range leading to one rupture event. We propose a simple way of constructing such a set of functions via piecewise linear approximation of the SMFS force vs time data and we prove the suitability of the approach both with synthetic data and experimentally. Additionally, when the spontaneous unfolding rate is the only unknown parameter, we find a correction factor that eliminates the bias of the ML estimator while also reducing its variance. Finally, we investigate which of several time-constrained experiment designs leads to better estimators.

Sample preparation for the quick sizing of metal nanoparticles by atomic force microscopy.

Two alternative pretreatment methods for depositing metal nanoparticles on mica for atomic force microscopy (AFM) imaging are presented. The treated substrates are flat and clean, thus they are amenable of use to characterize very small nanoparticles. The methods do not require any instrumentation or particular expertise. As they are also very quick, the need for storage of the prepared substrates is avoided altogether. These proposed methods, which are compared with the results of transmission electron microscopy analysis, allow the quick sizing and characterization of nanoparticles with the atomic force microscope and could thus help expanding the user community of nanoparticle researchers who could use the AFM for their characterization needs.

Screen-printed electrodes based on carbon nanotubes and cytochrome P450scc for highly sensitive cholesterol biosensors.

This paper is concerned with an investigation of electron transfer between cytochrome P450scc (CYP11A1) immobilized on nanostructured rhodium-graphite electrodes. Multi-walled carbon nanotubes (MWCNT) were deposited onto the rhodium-graphite electrodes by drop casting. Cytochrome P450scc was deposited onto MWCNT-modified rhodium-graphite electrodes. Cytochrome P450scc was also deposited onto both gold nanoparticle-modified and bare rhodium-graphite electrodes, in order to have a comparison with our previous works in this field. Cyclic voltammetry indicated largest enhanced activity of the enzyme at the MWCNT-modified surface. The role of the nanotubes in mediating electron transfer to the cytochrome P450scc was verified as further improved with respect to the case of rhodium-graphite electrodes modified by the use of gold nanoparticles. The sensitivity of our system in cholesterol sensing is higher by orders of magnitude with respect to other similar systems very recently published that are based on cholesterol oxidase and esterase. The electron transfer improvement attained by the use of MWCNT in P450-based cholesterol biosensors was demonstrated to be larger than 2.4 times with respect to the use of gold nanoparticles and 17.8 times larger with respect to the case of simple bare electrodes. The sensitivity was equal to 1.12 microA/(mM mm(2)) and the linearity of the biosensor response was improved with respect to the use of gold nanoparticles.

The reaction of alpha-synuclein with tyrosinase: possible implications for Parkinson disease.

Oxidative stress appears to be directly involved in the pathogenesis of Parkinson disease. Several different pathways have been identified for the production of oxidative stress conditions in nigral dopaminergic neurons, including a pathological accumulation of cytosolic dopamine with the subsequent production of toxic reactive oxygen species or the formation of highly reactive quinone species. On these premises, tyrosinase, a key copper enzyme known for its role in the synthesis of melanin in skin and hair, has been proposed to take part in the oxidative chemistry related to Parkinson disease. A study is herein presented of the in vitro reactivity of tyrosinase with alpha-synuclein, aimed at defining the molecular basis of their synergistic toxic effect. The results presented here indicate that, in conformity with the stringent specificity of tyrosinase, the exposed tyrosine side-chains are the reactive centers of alpha-synuclein. The reactivity of alpha-synuclein depends on whether it is free or membrane bound, and the chemical modifications on the tyrosinase-treated alpha-synuclein strongly influence its aggregation properties. On the basis of our results, we propose a cytotoxic model which includes a possible new toxic role for alpha-synuclein exacerbated by its direct chemical modification by tyrosinase.

Conformational equilibria in monomeric alpha-synuclein at the single-molecule level.

Human alpha-Synuclein (alphaSyn) 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 alphaSyn aggregation and formation of fibrils. Traditional bulk techniques used so far to solve this problem point to a direct correlation between alphaSyn's 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 alphaSyn. The conformational heterogeneity of monomeric alphaSyn was characterized at the single-molecule level. Three main classes of conformations, including disordered and "beta-like" structures, were directly observed and quantified without any interference from oligomeric soluble forms. The relative abundance of the "beta-like" structures significantly increased in different conditions promoting the aggregation of alphaSyn: 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 alphaSyn 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 alphaSyn.

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.