Chasing the elusive "in-between" state of the copper-amyloid β complex by X-ray absorption through partial thermal relaxation after photoreduction

The redox activity of Cu ions bound to the amyloid-β (Aβ) peptide is implicated as a source of oxidative stress in the context of Alzheimer's disease. In order to explain the efficient redox cycling between CuII -Aβ (distorted square-pyramidal) and CuI -Aβ (digonal) resting states, the existence of a low-populated "in-between" state, prone to bind Cu in both oxidation states, has been postulated. Here, we exploited the partial X-ray induced photoreduction at 10 K, followed by a thermal relaxation at 200 K, to trap and characterize by X-ray Absorption Spectroscopy (XAS) a partially reduced Cu-Aβ1-16 species different from the resting states. Remarkably, the XAS spectrum is well-fitted by a previously proposed model of the "in-between" state, hence providing the first direct spectroscopic characterization of an intermediate state. The present approach could be used to explore and identify the catalytic intermediates of other relevant metal complexes

Modelling Protein Plasticity: The Example of Frataxin and Its Variants

Frataxin (FXN) is a protein involved in storage and delivery of iron in the mitochondria. Single-point mutations in the FXN gene lead to reduced production of functional frataxin, with the consequent dyshomeostasis of iron. FXN variants are at the basis of neurological impairment (the Friedreich’s ataxia) and several types of cancer. By using altruistic metadynamics in conjunction with the maximal constrained entropy principle, we estimate the change of free energy in the protein unfolding of frataxin and of some of its pathological mutants. The sampled configurations highlight differences between the wild-type and mutated sequences in the stability of the folded state. In partial agreement with thermodynamic experiments, where most of the analyzed variants are characterized by lower thermal stability compared to wild type, the D104G variant is found with a stability comparable to the wild-type sequence and a lower water-accessible surface area. These observations, obtained with the new approach we propose in our work, point to a functional switch, affected by single-point mutations, of frataxin from iron storage to iron release. The method is suitable to investigate wide structural changes in proteins in general, after a proper tuning of the chosen collective variable used to perform the transition

Characterization of Pathology

The recent advances in imaging- and epilepsy-surgery techniques contributed critically to gain further insights in the structural brain abnormalities in patients with epilepsy, by combining traditional routine histopathological methods with functional analysis. This approach, together with the recent developments in the molecular genetics of epilepsies, has improved diagnosis and classification of hippocampal sclerosis (HS) and focal cortical dysplasias (FCD). However, the mechanisms underlying the development of specific structural abnormalities and their causal relationship with seizure development is still controversial, and requires appropriate animal models that recreate faithfully the key neuropathological features. The present chapter provides a detailed description of the most common histopathological findings encountered in epilepsy surgery patients with HS and FCD. The histopathological differences and similarities between the available animal models and the human equivalent are addressed, and the limitations of the animal models in mimicking the full range of neuropathological features of human disorders are discussed. Experimental details on the models can be found in separate book chapters