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Optical tweezers, bendable microneedles, and scanning force microscope probes make it possible to play with individual molecules of DNA, to stretch them beyond their natural length, to unzip and pull apart their strands (see schematic diagram), and to dissect them to create new molecules in situ. Depending on the method of measurement, the mechanical force necessary to separate the strands was in the range of 10-50 pN per base pair.

Label-free DNA detection plays a crucial role in developing point-of-care biochips. Capacitance detection is a promising technology for label-free detection. However, data published in literature often show evident time drift, large standard deviation, scattered data points, and poor reproducibility. To address these problems, mercapto-hexanol or similar alkanethiols are usually considered as blocking agents. The aim of the present paper is to investigate new blocking agents to further improve DNA probe surfaces. Data from AFM, SPR, florescence microscopy, and capacitance measurements are used to investigate new lipoate and ethylene-glycol molecules. The new surfaces offer further improvements in terms of diminished detection errors. Film structures are investigated at the nano-scale to justify the detection improvements in terms of probe surface quality. This study demonstrates the superiority of lipoate and ethylene-glycol molecules as blocking candidates when immobilizing molecular probes onto spot surfaces in label-free DNA biochip.

The recombinant production of a novel chimeric polyprotein is described. The new protein contains either wild-type beta(2)-microglobulin (beta(2)m) or its truncated variant (DeltaN6 beta(2)m) (see picture). Structural characterization is achieved by means of single-molecule force spectroscopy studies of specific beta(2)m regions which could be involved in amyloidogenesis.

This paper investigates technological advancements in developing reliable technologies for label free detection of cancer markers. Capacitance detection was used successfully to develop an immunosensor. Rigorous probe immobilization was implemented to obtain reliable capacitance measurements. The commonly considered alkanethiols for proteins immobilization do not allow sufficiently stable capacitance measurements. Therefore, new ethylene-glycol functionalized alkanethiols were used as more suitable materials to develop lab-on-a-chip detection of cancer markers. Results from AFM, Chemi-luminescence, Fluorescence, Cyclic Voltammetry, QCM, and capacitance measurements showed that three (ethylene-glycol) alkanethiols highly stabilized the sensing surfaces in time. The measured capacitance on the antibody layers was in the range of 60–70 nF/cm2 and 80–115 nF/cm2 for the antigen detection. The developed system was utilized to detect the Hepatocarcinoma marker SCCA, and the whole IgM fraction from Hepatocarcinoma patient’s serum. Electrode by electrode reproducibility within the same chip increased by one order of magnitude (variations were reduced from 30–40% down to less than 2%) using these new alkanethiols, while the reached limit in sensitivity was close to 2.43 μg/ml.

Improved label-free capacitive detection was very recently demonstrated for both ssDNA and antigen target molecules. That improvement is due to ethylene-glycol alkanethiols monolayers used as linkers for anchoring probe molecules. The presence of ethylene-glycol chains into the thin films greatly reduces time drift, detection error, and non-specific signals. It also increases ideality of the electrochemical interface behavior. However, the ideality of probe surfaces is partially lost when dealing with antibodies. The aim of this paper is to show this loss of ideality by means of capacitance measurements upon the frequency as acquired in label-free detection of cancer markers.

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 α-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 α-synuclein. The reactivity of α-synuclein depends on whether it is free or membrane bound, and the chemical modifications on the tyrosinase-treated α-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 α-synuclein exacerbated by its direct chemical modification by tyrosinase.

Statistical analysis of the profiles of palindromic DNA molecules imaged by atomic force microscopy provided evidence of the capability of a mica surface of recognizing DNA sequences. The differential free energy involved in this collective recognition process was estimated. A tentative model based on sequence-dependent imbalances of the electrostatic field normal to the surface (E) along the chain is proposed.

An increasing number of inter- and intramolecular interactions can nowadays be probed using single-molecule manipulation techniques. Protein unfolding and refolding is the most representative--though complex--of these interactions. Herein, we review the main modes of performing a force unfolding experiment: the velocity clamp and the new force clamp mode. We also compare some of the physical aspects behind the two most frequently used single-molecule manipulation instrumentations: optical tweezers and atomic force microscopes.

Nowadays, the development of experimental procedures for the determination of the secondary structure of RNA molecules is taking advantage of the novel single-molecule probing and imaging techniques. We report a method for the mapping of the secondary structure of RNA molecules spread on a flat surface by means of the atomic force microscope. Globular domains comprising groups of RNA secondary and tertiary structure elements separated by unstructured domains can be discerned in the micrographs and their position along the molecule contour can be measured directly on unstained specimens. We have analyzed the morphology of a population of single molecules of 3' fragments of the Turnip Yellow Mosaic Virus RNA shorter than 1 kb in different temperature and electrolytic conditions. We found a satisfying agreement of the shape of the imaged structures with previously available evidence. The method we have developed can be used to map also different types of RNA molecules and has the advantage of showing the distribution of the single molecule conformations within the population.

A mechanochemical study of the process of adhesion of plasma proteins to the surface of dialysis membranes was carried out with a scanning force microscope (SFM) in the force spectroscopy mode. Three representative blood plasma proteins (fibronectin, fibrinogen, and albumin) covalently were grafted to a SFM probe, and the adhesion forces of these proteins to cellulosic and synthetic dialysis membranes were measured. The experiment was tailored to apply a controlled load on the protein molecules adsorbed onto the surface in order to simulate the squeezing forces exerted on them during blood filtration. The de-adhesion forces, measured using this new approach for studying the interaction between a protein and dialysis membranes, suggest that the membrane's topography, at a nanometer scale, plays a critical role in the adhesion process. This result was strongly supported by parallel experiments performed on a flattened glass surface with the same dominant hydrophilic character as dialysis membranes. In contrast, a hydrophobic polystyrene surface led to de-adhesion forces at least one order of magnitude greater, overwhelming any possible shape recognition process between the protein molecules and the surface.

Most of the scanning force microscopy (SFM) images of supercoiled DNA on untreated mica thus far reported have not shown tight plectonemic structure seen by electron microscopy, but instead less coiled molecules and sometimes a partly "condensed" state with intimate chain-chain interactions. By observing time-lapse images of conformational changes of DNA induced by decreasing ionic strength of imaging buffer in solution SFM, we could show that the process of water rinsing, an indispensable step for preparation of dried samples, may be responsible for some of the conformational anomalies in the images previously reported. We have studied several protocols to observe supercoiled DNA molecules by SFM and discuss the merits and the demerits. Images obtained following uranyl acetate treatment may be ideal for the detection of DNA damage, as the supercoiled and nicked forms are easily distinguishable.

Familial and idiopathic Parkinson's disease (PD) is associated with the abnormal neuronal accumulation of alpha-synuclein (aS) leading to beta-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 14-3-3 eta 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 14-3-3 eta and aS interact in PD brains remain unclear. Herein, we show that while 14-3-3 eta 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 14-3-3 eta is present in sub-stoichiometric amounts relative to aS. When aS level is overwhelmingly higher than that of 14-3-3 eta, the fibrillation process becomes a sequestration mechanism for 14-3-3 eta, undermining all processes governed by this protein. Using a panel of complementary techniques, we single out the stage of aggregation at which the aS/14-3-3 eta 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/14-3-3 eta interaction and in understanding the critical aggregation step at which 14-3-3 eta has the potential to rescue aS-induced cellular toxicity.

Cationic block copolymers, consisting of a poly(ethylene glycol) block and a block deriving from the poly(dimethylamino)ethyl methacrylate were prepared via a two-step procedure, based on the use of macroinitiators. By appropriately changing the experimental conditions and reacting the poly(dimethylamino)ethyl methacrylate block with iodo- or bromo-alkyl derivatives, a variety of ionic block copolymers with tuned physicochemical properties were prepared. These block copolymers are able to spontaneously self-assemble with plasmid DNA to produce oriented and shielded vectors, with physicochemical properties appropriate for in vivo applications. In addition, the formation of a complex between the cationic block copolymer and the plasmid DNA results in a nuclease resistance increase due to the stable nature of the complex.

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. Microsc. Res. Tech., 2008. © 2008 Wiley-Liss, Inc.

Polynucleotide: adenosine glycosidases (PNAG) are a class of plant and bacterial enzymes commonly known as ribosome-inactivating proteins (RIP). They are presently classified as rRNA N-glycosidases in the enzyme nomenclature [EC 3.2.2.22]. Several activities on nucleic acids, other than depurination, have been attributed to PNAG: in particular modifications induced in circular plasmids, including linearisation and topological changes, and cleavage of guanidinic residues. Here we describe a chromatographic procedure to obtain nuclease-free PNAG by dye-chromatography onto Procion Red derivatized Sepharose((R)). Highly purified enzymes depurinate extensively pBR322 circular, supercoiled DNA at neutral pH and exhibit neither DNase nor DNA glycolyase activities, do not cause topological changes, and adenine is the only base released from DNA and rRNA, even at very high enzyme concentrations. A scanning force microscopy (SFM) study of pBR322 treated with saporin-S6 confirmed that (i) this PNAG binds extensively to the plasmid, (ii) the distribution of the bound saporin-S6 molecules along the DNA chain is markedly variable, (iii) plasmids already digested with saporin-S6 do not appear fragmented or topologically modified. The observations here described demonstrate that polynucleotide:adenosine glycosidase is the sole enzymatic activity of the four ribosome-inactivating proteins gelonin, momordin I, pokeweed antiviral protein from seeds and saporin-S6. These proteins belong to different families, suggesting that the findings here described may be generalized to all PNAG.

Isolation of a His-tagged protein by means of a Ni - nitrilotriacetate-functionalized chromatographic matrix or biosensor was simulated in a single-molecule experiment, and the mechanochemistry-that is, the interplay between mechanical and chemical forces (shown schematically)-was studied with a scanning force microscope.

A large improvement of the electrochemical capacitive detection of ssDNA and antigens by using ethylene-glycol alkanethiol monolayers has been recently demonstrated. Alkanethiol monolayers without ethylene-glycol functionalization do not show the required electrochemical stability. In this paper, we demonstrate that both phenomena are related to their morphological structure at nano-scale. Ethylene-glycol monolayers were compared with similar films without ethylene-glycol function. Capacitance measurements were used to verify the improved capacitance stability of the electrochemical interface. AFM images were used to investigate films packing and morphology at the nano-scale. This letter demonstrates that films without ethylene-glycol segment present deep grooves which are related to electrochemical instability while the improvement in case of ethylene-glycols is due to the absence of such a feature. (C) 2009 Elsevier B.V. All rights reserved.

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. Contact: massimo.sandal@unibo.it

Optische Pinzetten, biegsame Mikronadeln und fur die Rasterkraftmikroskopie geeignete Praparate ermoglichen es, mit einzelnen DNA-Molekulen zu spielen: Diese konnen uber ihre normale Lange gestreckt, ihre beiden Strange wie ein Reisverschlus geoffnet (im Bild schematisch dargestellt) und vollstandig voneinander getrennt sowie zerschnitten werden, wodurch in situ neuartige Molekulkonformationen entstehen. Die mechanischen Krafte fur das Aufbrechen der Doppelstrange liegen je nach Mesmethode im Bereich von 10–50 pN pro Basenpaar.

The use of the scanning force microscope (SFM) to visualize and analyze chromatin fiber structures is presented. Protocols to prepare chromatin fibers for SFM imaging of fibers in air and in buffer are first discussed. Next, the conditions for acquiring high-quality SFM images such as optimal instrumental parameters, appropriate deposition substrates, and adequate procedures of sample deposition are described. It is shown that analysis and quantitation of the SFM images support an irregular, three-dimensional arrangement of nucleosomes in the native chromatin fiber. This structure is lost in linker histone-depleted fibers, which show, instead, a beads-on-a-string structure. Molecular modeling of the chromatin fiber structures and computer simulation of the SFM imaging process indicate that the natural variability of the linker length may be the major determinant of the structural irregularity of the native chromatin fiber. Removal of linker histones (H1/H5) may change the amount of DNA wrapped around the histone octamer, which in turn may induce the transition from a three-dimensional irregular helix to an extended beads-on-a-string structure. Studies of trinucleosomes indicate that both the average successive nucleosome center-to-center distance and the average angle between two successive linkers increase upon the removal of linker histone.

Quercetin and other flavonoids which had been previously found to be mutagenic in the Ames test, have been proved to inhibit tumor development in several experimental animal models. We studied the interaction between DNA and a series of flavonoids whose biological activity was known to span a wide range of potency: quercetin (the most active), morin, rutin, naringin, and 2,3-dihydroquercetin (inactive). The sensitivity of equilibrium binding analysis carried out by the dextran-polyethylene glycol-H 2 O biphase system was not high enough to reveal and evaluate the binding affinity of these flavonoids toward DNA. The much more sensitive flow linear dichroism technique provided evidence that they can bind DNA by intercalation and that their affinity for DNA increased following the same sequence of their biological activity

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.

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.

The aggregation of alpha-synuclein into amyloid fibrils constitutes a key step in the onset of Parkinson's disease. Amyloid fibrils of alpha-synuclein are the major component of Lewy bodies, histological hallmarks of the disease. Little is known about the mechanism of aggregation of alpha-synuclein. During this process, alpha-synuclein forms transient intermediates that are considered to be toxic species. The dimerization of alpha-synuclein could represent a rate-limiting step in the aggregation of the protein. Here, we analyzed four covalent dimers of alpha-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 alpha-helical transition upon interaction with SDS. All protein species are able to form amyloid-like fibrils. The reciprocal orientation of the alpha-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 alpha-synuclein fibrils. This study demonstrates that although alpha-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. Citation: Pivato M, De Franceschi G, Tosatto L, Frare E, Kumar D, et al. (2012) Covalent alpha-Synuclein Dimers: Chemico-Physical and Aggregation Properties. PLoS ONE 7(12): e50027. doi:10.1371/journal.pone.0050027

Unfixed chicken erythrocyte chromatin fibers in very low salt have been imaged with a scanning force microscope operating in the tapping mode in air at ambient humidity. These images reveal a three-dimensional organization of the fibers. The planar "zig-zag" conformation is rare, and extended "beads-on-a-string" fibers are seen only in chromatin depleted of histones H1 and H5. Glutaraldehyde fixation reveals very similar structures. Fibers fixed in 10 mM salt appear somewhat more compacted. These results, when compared with modeling studies, suggest that chromatin fibers may exist as irregular three-dimensional arrays of nucleosomes even at low ionic strength.

Calcitonin (CT) inhibits osteoclastic bone resorption and induces calcium uptake from body fluids. A comparative study of the conformational behaviours of therapeutic calcitonins [salmon (s), eel (e), a synthetic eel calcitonin analogue (Elcatonin), porcine (p) and human (h) calcitonins] as a function of solvent polarity and temperature have been performed by circular dichroism spectroscopy. Elements of secondary structure were lacking in H2O but could be observed in 2,2,2-trifluoroethanol and sodium dodecyl sulphate. In particular, similar amounts of alpha-helical content (four alpha-helical turns) were estimated in trifluoroethanol despite the considerable differences in amino acid sequences. The relative ability to form an alpha helix, assessed by trifluoroethanol/H2O titration, was found to be Elcatonin > sCT > pCT > eCT > hCT. In Elcatonin, sCT, pCT and eCT the four alpha-helical turns were promoted almost completely in a single step, between 0 and 35% trifluoroethanol, unlike hCT where helical structure formation has been reported to involve two steps over the whole trifluoroethanol/H2O range [Arvinte, T. & Drake, A. F. (1993) J. Biol. Chem. 268, 6408-6414]. In SDS, which mimics the membrane environment, conformational differences (3-4 helical turns in Elcatonin, sCT, eCT versus one helical turn in pCT, hCT) were observed and correlate well with biological activity (Elcatonin = sCT = eCT > pCT = hCT). Low-temperature studies in a cryogenic solvent mixture showed the formation of high alpha-helix content (similar to that in trifluoroethanol) in Elcatonin, sCT, eCT and pCT, whilst a left-handed extended helix (3(1) helix) was formed in hCT. This is consistent with the hypothesis of 'linear' and 'helical' calcitonin receptors [Nakanuta, H., Orlowski, R. C. & Epand, R. M. (1990) Endocrinology 127, 163-169].

Human angiostatin 1 ± 5 [Ang(1 ± 5)], a 57 kDa proteolitic fragment of human plasminogen, consists of five compact globular modules called Kringle domains with very similar gross architecture and remarkable sequence homology. They are built around a hydrophobic core and exhibit a triple-loop topology defined by three internal disulfide bonds: cys1 ± cys80 (1), cys22 ± cys63 (2), and cys51 ± cys75 (3) (Figure 1a).[1] Here we report on a single-molecule force-spectroscopy study that shows how the redox environment can control the topology and mechanical properties of angiostatin by modifying the extent of pairing of the internal disulfide bonds. Ang(1 ± 5) molecules were deposited on a polystyrene surface; the tip of a scanning force microscope (SFM) was brought into contact with this surface and retracted after reaching a force of 1 nN. This cycle was repeated many hundreds of times, first under phosphate buffer saline (PBS), Figure 1. a) Schematic representation of the Ang(1 ± 5) structure with the triple-loop topology of its five Kringle domains (K1 ±K5), determined in each case by a sequence of three disulfide bonds: cys1 ± cys80 (1), cys22 ± cys63 (2), and cys51 ± cys75 (3). The two asterisks (*) indicate the position of an additional, inter-Kringle disulfide bond. b) Pathway of the sequential reduction of the most exposed (1) and then of the more interior (2) and (3) bonds. This reduction pathway leads to gradually lengthening topological loops (red) that are exposed to the force exerted by the SFM tip at the two ends of the Kringle domain.

Protecting osmolytes play a crucial role in preventing protein denaturation in harsh environmental conditions of living organisms. Experimental evidence is provided for a mechanism of protein-fold stabilization by these molecules that is in accord with the hypothesis of a backbone-based osmophobic effect. (In picture: ”G=free energy, [O]=osmolyte concentration, Ç=unfolding reaction coordinate.)

The rearrangement of allyl p-dimethylaminobenzenesulphonate (ASE) to form a zwitterionic product has already been recognized as an effective probe for the study of reactivity within the smectic B phase [4, 5, 19]. We have used deuterium NMR, linear dichroism and X-ray diffraction techniques to investigate the phase diagram of the ASE-OS35 reaction system. The partitioning of the reactant molecules between coexisting smectic, nematic and/or isotropic phases and the structural organization of the smectic catalytic host at different temperatures and reactant guest concentrations have been characterized. On the basis of these measurements, a model of ASE reactivity in smectic solvents has been developed. The reaction takes place provided that coexisting isotropic or nematic phases are present to act as a reservoir for the ASE reactant molecules prior to their entering the smectic phase; they then react and leave the smectic phase as a zwitterionic product. The analogy between this model of reactivity...

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; © 2011 Wiley-Liss, Inc.

This chapter contains sections titled: Abstract Introduction Basic Elements of the SMFS Methodology for Protein Folding Studies the SMFS Methodology in the Studies of the Mechanical Properties and Interactions of IDPs Probing the Conformational Equilibria of the IDPs Involved in Neurodegenerative Diseases: the Case of α-Synuclein Future Perspectives References

DNA fragments can be immobilized by Langmuir - Blodgett films of 1-hexadecylamine. The inclusion in these LB films of 5′-GTAAAACGACGGCCAGT-3′ was proved by a radioactive technique. An X-ray diffraction study proved an overall lamellar ordering of the samples and suggested a structural organization in which layers of oligonucleotide molecules are sandwiched between two monolayers of 1-hexadecylamine. These monolayers are likely to turn their amino groups surfaces to the oligonucleotide molecules as to screen their anionic charges.

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.

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.

Label-free DNA detection is of crucial role to when developing point-of-care biochips to be used in personalized therapy. Capacitance detection is a promising technology for label-free DNA detection. However, data published in literature often show evident time drift, large standard deviation, scattered data points, and poor reproducibility. To solve these problems, alkanethiol molecules such as mercapto-hexanol are usually considered as blocking agents. The aim of the present paper is to investigate new blocking agents to further improve DNA probe surfaces. Data from AFM, SPR, fluorescence microscopy, and capacitance measurements are used to demonstrate the new lipoates molecules. Moreover precursor layers obtained by using Ethylene-glycol alkanethiols offer further improvements in terms of diminished detection errors. Film structure is investigated at the nano-scale to justify the detection improvements in terms of probe surface quality. This study demonstrates the superiority of lipoate and Ethylene-glycol molecules as blocking candidates when immobilizing molecular probes onto spot surfaces in label-free DNA biochip.

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. © 2009 American Institute of Physics.