Jacopo Vertemara

Intrinsically disordered proteins (IDPs) and regions constitute a significant fraction of the proteome. Unlike globular proteins, IDPs exist as dynamic conformational ensembles, allowing functional versatility while challenging conventional structural methods, necessitating an integrated, multi-method approach. Here, we systematically compare IDPs and globular proteins using Fourier Transform Infrared (FTIR) and circular dichroism (CD) spectroscopies to investigate not simply the presence of disorder but also its structural organization. By analyzing the second derivatives of the Amide I′ absorption spectra, we identified a distinct spectral fingerprint of IDPs. Detailed analysis reveals that the spectral features encode distinct modes of structural organization within conformationally heterogeneous ensembles. Notably, the integration of second-derivative FTIR (SD-IR) and CD spectroscopies uncovers conformational states that are not captured by conventional techniques devoted to secondary structure characterization. Specifically, we identified conditions in which proteins retain a conformational order despite exhibiting random coil CD signatures (“ordered random coils”) as well as states displaying canonical α-helical CD features while maintaining substantial structural heterogeneity (“disordered α-helices”). Molecular dynamics simulations support these assignments, revealing the structural basis underlying the spectroscopic findings. Finally, we demonstrate the efficacy of SD-IR spectroscopy for validating putative IDPs, exemplified by the silkworm protein sericin. Overall, this study establishes second-derivative analysis of the Amide I′ band not merely as a diagnostic marker of disorder but also as a powerful approach to resolve distinct organizational states within conformationally heterogeneous protein ensembles.

The performance of artificial molecular machines relies on the interplay between molecular design and environmental factors, yet how solvation shapes their energy landscapes and kinetics remains poorly understood. Here, we combine well-tempered and infrequent metadynamics to investigate equilibrium shuttling in a minimal [2]rotaxane inspired by Borsley's fuel-driven molecular motor. By systematically varying solvent polarity and hydrogen-bonding capacity, we uncover distinct thermodynamic and kinetic regimes that govern macrocycle motion. In highly polar, hydrogen-bond-accepting media, the macrocycle adopts a symmetric distribution between binding sites, with enthalpic and entropic forces in direct competition. Conversely, in low-polarity, hydrogen-bond-donating environments, the axle undergoes a conformational collapse that entropically biases occupancy toward a single station in the absence of chemical fuel. Despite comparable free-energy barriers across conditions (9-13 kcal/mol), the transition pathways exhibit pronounced solvent-dependent asymmetries and energetic ruggedness. These findings provide a molecular-level framework for understanding how solvation dictates passive ratchet behavior and offer strategic insights for designing high-performance molecular machines tailored to complex media.

Thiol-containing drugs may interact with a region of tropomyosin receptor kinase A (TrkA), potentially inhibiting its activation by nerve growth factor (NGF). This action has been linked to potential analgesic activities. Here, we describe the ability of erdosteine, a thiolic compound classified as a mucolytic agent, to bind to the TrkA receptor sequence in silico and its in vitro effects on TrkA activation induced by NGF in cultured human neuroblastoma cells. Our results show that erdosteine and its metabolite, Met-1, bind to the TrkA receptor pocket, involving the primary TrkA residues Glu331, Arg347, His298, and His297. Furthermore, Met-1 has the ability to reduce the disulfide bridge between Cys300 and Cys345 of TrkA. In vitro measurement of TrkA autophosphorylation following NGF activation confirmed that erdosteine and Met-1 interfere with NGF-induced TrkA activation, leading to a consequent loss of the molecular recognition and spatial reorganization necessary for the induction of the autophosphorylation process. This effect was inhibited by low millimolar concentrations of the two compounds, reaching a maximal inhibition (around 40%) after 24 h of exposure to 1 mM erdosteine, and then plateauing. These findings suggest that erdosteine can act as a TrkA antagonist, thus indicating that this drug may have potential as an analgesic via a novel non-opioid mechanism of action operating through NGF signaling inhibition at the level of TrkA.

Societal Impact Statement: The legume crop cowpea is grown worldwide, but 90% of the world's total share is produced in Africa. It is a promising species due to its resilience properties, balance of macro and micronutrients and presence of health‐promoting bioactive compounds. In African countries, cowpea has a crucial role in guaranteeing food security as a subsistence crop for families and commercial income for small farmers. The discovery of compounds with high nutraceutical value and bioactive properties supports socio‐economic policies to improve health and nutrition, especially in low‐ and middle‐income countries. In turn, this encourages biodiversity protection and crop enhancement programmes.Summary: Bowman–Birk protease inhibitors (BBIs) are a restricted group of small proteins in plants mainly involved in defence mechanisms against pests. BBIs are demonstrated to be active components capable of reducing the viabilities of different cancer cell lines. BBI bioactivity is directly linked to the inhibition capacity, but the variability and the efficiency against the physiological targets of different BBI isoforms remain still unexplored. We analysed the natural genetic diversity of two main genes encoding BBI trypsin‐trypsin (BBI‐TT) and trypsin‐chymotrypsin (BBI‐TC) in wild and domesticated cowpea samples mainly spread in Sub‐Saharan Africa. We analysed DNA sequences and respective amino acidic isoforms/isoproteins to explore signs of natural selection and haplotype relationships. Moreover, we calculated the binding energy between BBIs and their biological targets to identify which are the most efficient inhibitors and their geographical locations. We found a high level of haplotype diversity for both genes, almost exclusively in wild accessions and detected positive and negative selection signals in the amino acid sequences. Furthermore, in the wild diversity pool, some BBI‐TT and BBI‐TC mature proteins were potentially better interactors with the physiological targets. The long interaction between plant‐pathogen has selected new and useful isoforms in wild lineages that have allowed the chances of survival of the species to improve. On the other hand, the domestication process has produced an intense bottleneck leaving only poorly efficient BBI variants. In addition to providing information on the natural diversity and evolution of BBIs, our work discusses the potential applications in agriculture and human health.

N-acetylcysteine (NAC), a mucolytic agent and an antidote to acetaminophen intoxication, has been studied in experimental conditions and trials exploring its analgesic activity based on its antioxidant and anti-inflammatory properties. The purpose of this study is to investigate additional mechanisms, namely, the inhibition of nerve growth factor (NGF) and the activation of the Tropomyosin receptor kinase A (TrkA) receptor, which is responsible for nociception. In silico studies were conducted to evaluate dithiothreitol and NAC’s interaction with TrkA. We also measured the autophosphorylation of TrkA in SH-SY5Y cells via ELISA to assess NAC’s in vitro activity against NGF-induced TrkA activation. The in silico and in vitro tests show that NAC interferes with NGF-induced TrkA activation. In particular, NAC breaks the disulfide-bound Cys 300–345 of TrkA, perturbing the NGF-TrkA interaction and producing a rearrangement of the binding site, inducing a consequent loss of their molecular recognition and spatial reorganization, which are necessary for the induction of the autophosphorylation process. The latter was inhibited by 40% using 20 mM NAC. These findings suggest that NAC could have a role as a TrkA antagonist, an action that may contribute to the activity and use of NAC in various pain states (acute, chronic, nociplastic) sustained by NGF hyperactivity and/or accompanied by spinal cord sensitization.

Flavodoxins are enzymes that contain the redox-active flavin mononucleotide (FMN) cofactor and play a crucial role in numerous biological processes, including energy conversion and electron transfer. Since the redox characteristics of flavodoxins are significantly impacted by the molecular environment of the FMN cofactor, the evaluation of the interplay between the redox properties of the flavin cofactor and its molecular surroundings in flavoproteins is a critical area of investigation for both fundamental research and technological advancements, as the electrochemical tuning of flavoproteins is necessary for optimal interaction with redox acceptor or donor molecules. In order to facilitate the rational design of biomolecular devices, it is imperative to have access to computational tools that can accurately predict the redox potential of both natural and artificial flavoproteins. In this study, we have investigated the feasibility of using non-equilibrium thermodynamic integration protocols to reliably predict the redox potential of flavodoxins. Using as a test set the wild-type flavodoxin from Clostridium Beijerinckii and eight experimentally characterized single-point mutants, we have computed their redox potential. Our results show that 75% (6 out of 8) of the calculated reaction free energies are within 1 kcal/mol of the experimental values, and none exceed an error of 2 kcal/mol, confirming that non-equilibrium thermodynamic integration is a trustworthy tool for the quantitative estimation of the redox potential of this biologically and technologically significant class of enzymes.

DNA double-strand breaks (DSBs) are a significant threat to cell viability due to the induction of genome instability and the potential loss of genetic information. One of the key players for early DNA damage response is the conserved Mre11/Rad50 Nbs1/Xrs2 (MRN/X) complex, which is quickly recruited to the DNA’s ruptured ends and is required for their tethering and their subsequent repair via different pathways. The MRN/X complex associates with several other proteins to exert its functions, but it also exploits sophisticated internal dynamic properties to orchestrate the several steps required to address the damage. In this review, we summarize the intrinsic molecular features of the MRN/X complex through biophysical, structural, and computational analyses in order to describe the conformational transitions that allow for this complex to accomplish its multiple functions.

Telomere biology disorders (TBDs) are characterized by short telomeres, premature aging, bone marrow failure and cancer predisposition. Germline mutations in NHP2, encoding for one component of the telomerase cofactor H/ACA RNA binding complex together with Dyskerin, NOP10 and GAR1, have been previously reported in rare cases of TBDs. Here, we report two novel NHP2 variants (NHP2-A39T and NHP2-T44M) identified in a compound heterozygous patient affected by premature aging, bone marrow failure/myelodysplastic syndrome and gastric cancer. Although still able to support cell viability, both variants reduce the levels of hTR, the telomerase RNA component, and telomerase activity, expanding the panel of NHP2 pathological variants. Furthermore, both variants fail to be incorporated in the H/ACA RNA binding complex when in competition with wild-type endogenous NHP2, and the lack of incorporation causes their drastic proteasomal degradation. By RoseTTAFold prediction followed by molecular dynamics simulations, we reveal a dramatic distortion of residues 33–41, which normally position on top of the NHP2 core, as the main defect of NHP2-A39T, and high flexibility and the misplacement of the N-terminal region (residues 1–24) in NHP2-T44M and, to a lower degree, in NHP2-A39T. Because deletion of amino acids 2–24 causes a reduction in NHP2 levels only in the presence of wild-type NHP2, while deletion of amino acids 2–38 completely disrupts NHP2 stability, we propose that the two variants are mis-incorporated into the H/ACA binding complex due to the altered dynamics of the first 23 amino acids and/or the distortion of the residues 25–41 loop.

Oxidative stress and metal dyshomeostasis are considered crucial factors in the pathogenesis of Alzheimer’s disease (AD). Indeed, transition metal ions such as Cu(II) can generate reactive oxygen species (ROS) via O₂ Fenton-like reduction, catalyzed by Cu(II) coordinated to the amyloid-beta (Aβ) peptide. Despite intensive efforts, the mechanisms of ROS-induced molecular damage remain poorly understood. In the present paper, we investigate, on the basis of Density Functional Theory (DFT) computations, a possible mechanism of the OH radical propagation toward membrane phospholipid polar head and fatty acid chains starting from the end-product of the OH radical generation by Cu(II)-Aβ. Using phosphatidylcholine as a model of a single unit inside a membrane, we evaluated the thermochemistry of the OH propagation with the oxidation of a C-H bond and the formation of the radical moiety. The DFT results show that Cu(II)-Aβ-OH can oxidize only sn-2 C-H bonds of the polar head and can easily oxidize the C-H bond adjacent to the carbon–carbon double bond in a mono or bis unsaturated fatty acid chain. These results are discussed on the basis of the recent literature on in vitro Aβ metal-catalyzed oxidation and on the possible implications in the AD oxidative stress mechanism.

Molecular modeling techniques have become indispensable in many fields of molecular sciences in which the details related to mechanisms and reactivity need to be studied at an atomistic level. This review article provides a collection of computational modeling works on a topic of enormous interest and urgent relevance: the properties of metalloenzymes involved in the degradation and valorization of natural biopolymers and synthetic plastics on the basis of both circular biofuel production and bioremediation strategies. In particular, we will focus on lytic polysaccharide monooxygenase, laccases, and various heme peroxidases involved in the processing of polysaccharides, lignins, rubbers, and some synthetic polymers. Special attention will be dedicated to the interaction between these enzymes and their substrate studied at different levels of theory, starting from classical molecular docking and molecular dynamics techniques up to techniques based on quantum chemistry.