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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.

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.

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.

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.

We present a set of DNA supramolecular architectures based on the polymerization of discrete DNA tiles having the shape of parallelograms and designed to have a one-dimensional inter-tile connectivity. Tiles bind to each other with two connections, which have different thermal stabilities. We discuss how this difference in stability implies that the same monomeric tile can yield supramolecular polymers of different shapes just by changing the polymerization conditions. We show how this system reacts to external stimuli by interconverting between some of its possible states. Concurrently, we show how performing the polymerization on a surface can influence its outcome.

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 nanometer scale is a special place where all sciences meet and develop a particularly strong interdisciplinarity. While biology is a source of inspiration for nanoscientists, chemistry has a central role in turning inspirations and methods from biological systems to nanotechnological use. DNA is the biological molecule by which nanoscience and nanotechnology is mostly fascinated. Nature uses DNA not only as a repository of the genetic information, but also as a controller of the expression of the genes it contains. Thus, there are codes embedded in the DNA sequence that serve to control recognition processes on the atomic scale, such as the base pairing, and others that control processes taking place on the nanoscale. From the chemical point of view, DNA is the supramolecular building block with the highest informational content. Nanoscience has therefore the opportunity of using DNA molecules to increase the level of complexity and efficiency in self-assembling and self-directing processes.

Scanning force microscopy was used to study in fluid the conformational fluctuations of two double-stranded DNA molecules resulting from differently cut pBR322 circular DNAs. A new approach was conceived to monitor the thermodynamic equilibrium of the chain dynamics on different scale lengths. This method made it possible to demonstrate that both the observed DNA molecules were allowed to equilibrate only on their local small-scale dynamics during the time of the experiment. This capability of monitoring the length scale and the time scale of the equilibration processes in the dynamics of a DNA chain is relevant to give an insight in the thermodynamics of the DNA binding with proteins and synthetic ligands. It was also shown that the small-scale equilibration of the DNA chain during surface-restricted dynamics is enough to allow a valid measurement of the local sequence-dependent curvature.

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., (© 1998 WILEY-VCH Verlag GmbH, Weinheim, Fed. Rep. of Germany.)