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

Abstract 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. Contact: [email protected] Supplementary information: Supplementary data are available at Bioinformatics online

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

Natively unstructured proteins defy the classical "one sequence-one structure" paradigm of protein science. Monomers of these proteins in pathological conditions can aggregate in the cell, a process that underlies socially relevant neurodegenerative diseases such as Alzheimer and Parkinson. A full comprehension of the formation and structure of the so-called misfolded intermediates from which the aggregated states ensue is still lacking. We characterized the folding and the conformational diversity of alpha-synuclein (aSyn), a natively unstructured protein involved in Parkinson disease, by mechanically stretching single molecules of this protein and recording their mechanical properties. These experiments permitted us to directly observe directly and quantify three main classes of conformations that, under in vitro physiological conditions, exist simultaneously in the aSyn sample, including disordered and "beta-like" structures. We found that this class of "beta-like" structures is directly related to aSyn aggregation. In fact, their relative abundance increases drastically in three different conditions known to promote the formation of aSyn fibrils: the presence of Cu2+, the occurrence of the pathogenic A30P mutation, and high ionic strength. We expect that a critical concentration of aSyn with a "beta-like" structure must be reached to trigger fibril formation. This critical concentration is therefore controlled by a chemical equilibrium. Novel pharmacological strategies can now be tailored to act upstream, before the aggregation process ensues, by targeting this equilibrium. To this end, Single Molecule Force Spectroscopy can be an effective tool to tailor and test new pharmacological agents.Comment: 37 pages, 9 figures (including supplementary material

The Interplay between Chemistry and Mechanics in the Transduction of a Mechanical Signal into a Biochemical Function

There are many processes in biology in which mechanical forces are generated. Force-bearing networks can transduce locally developed mechanical signals very extensively over different parts of the cell or tissues. In this article we conduct an overview of this kind of mechanical transduction, focusing in particular on the multiple layers of complexity displayed by the mechanisms that control and trigger the conversion of a mechanical signal into a biochemical function. Single molecule methodologies, through their capability to introduce the force in studies of biological processes in which mechanical stresses are developed, are unveiling subtle intertwining mechanisms between chemistry and mechanics and in particular are revealing how chemistry can control mechanics. The possibility that chemistry interplays with mechanics should be always considered in biochemical studies.Comment: 50 pages, 18 figure

Loss of Capacitance Ideality in Label-Free Immuno-Chip

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

Improving Probe Immobilization for Label-Free Capacitive Detection of DNA Hybridization on Microfabricated Gold Electrodes

Alternative approaches to labeled optical detection for DNA arrays are actively investigated for low-cost point-of-care applications. In this domain, label-free capacitive detection is one of the most intensely studied techniques. It is based on the idea to detect the Helmholtz ion layer displacements when molecular recognition occurs at the electrodes/solution interface. The sensing layer is usually prepared by using thiols terminated DNA single-strength oligonucleotide probes on top of the sensor electrodes. However, published data shows evident time drift, which greatly complicates signal conditioning and processing and ultimately increases the uncertainty in DNA recognition sensing. The aim of this work is to show that newly developed ethylene-glycol functionalized alkanethiols greatly reduce time drift, thereby significantly improving capacitance based label-free detection of DNA

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

Complex associates of plasmid DNA and a novel class of block copolymers with PEG and cationic segments as new vectors for gene delivery

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

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

SIMPLE SUMMARY: Prion diseases are neurodegenerative disorders caused by the amyloidal aggregation of the cellular prion protein. We apply single-molecule force spectroscopy approaches to study the unfolding of prion protein monomers and dimers in different orientations. We find heterogeneous behavior in the prion protein unfolding and an interesting difference between the dimer orientations whereby the dimer in which the C-termini are joined unfolds at a higher force, implying a more stable structure owing to interactions between the C-termini. These results may contribute to a better understanding of the initial steps of oligomer assembly during prion diseases. ABSTRACT: 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 (PrP(C)) into amyloidal forms denoted as prions or PrP(Sc). 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

Interface Layering Phenomena in Capacitance Detection of DNA with Biochips

Reliable DNA detection is of great importance for the development of the Lab-on-chip technology. The effort of the most recent projects on this field is to integrate all necessary operations, such as sample preparation (mixing, PCR amplification) together with the sensor user for DNA detection. Among the different ways to sense the DNA hybridization, fluorescence based detection has been favored by the market. However, fluorescence based approaches require that the DNA targets are labeled by means of chromophores. As an alternative label-free DNA detection method, capacitance detection was recently proposed by different authors. While this effect has been successfully demonstrated by several groups, the model used for data analysis is far too simple to describe the real behavior of a DNA sensor. The aim of the present paper is to propose a different electrochemical model to describe DNA capacitance detection