Stefania Brocca, Erika Ponzini, Carlo Santambrogio, Rita Grandori
One of the challenges of modern structural biology is to account for protein structural dynamics. Intrinsically disordered proteins (IDPs), which do not fold spontaneously into a defined conformation, existing, instead, as highly heterogeneous conformational ensembles, provide an extreme and challenging example of protein dynamics. Native mass spectrometry (nMS) allows dissecting the molecular population in its main conformational components of different compactness, assessing structural parameters, covalent modifications, and binding states for each resolved species. This review summarizes the work performed by our research group in investigating the conformational states of human α-synuclein (α-syn), an IDP involved in Parkinson’s disease (PD) and other neurodegenerative conditions, by applying nMS. This case study is taken as an example, highlighting the complementarity of nMS to other biophysical techniques, such as circular dichroism (CD), Fourier-transform infrared (FTIR) spectroscopy, nuclear magnetic resonance (NMR), atomic force microscopy (AFM), and molecular-dynamics (MD) simulations. This multi-technological approach is useful in analyzing the response of α-syn conformational ensembles to covalent modifications and noncovalent interactions with metal ions, small ligands, and other proteins.
Models of fibrosis are largely molecular and cell-centred. How whole-organism load geometry constrains long-term tissue fate is comparatively underspecified. The Fibrosis–Plasticity Axis (FPA), a conceptual continuum from reversible remodelling capacity to structural fixation, is proposed here as an organising frame; the physical steps linking load geometry to position on that continuum have not been articulated as a testable chain. To formulate, and render falsifiable, the hypothesis that deformation-generated electrical phenomena in collagen-rich matrix act as a load-geometry-dependent modulator of remodelling, and to define the constructs required to test it. We synthesise evidence from tissue electromechanics, mechanotransduction and connective-tissue physiology, and grade each proposed causal link by evidence level. We introduce, as constructs original to this work: the Geometric Origin of the Body (GOB), the equilibrium configuration around which load-bearing architecture is organised; the Geometric Optimal Pressure Load (GOPeL), the hypothesised load distribution permitting maximal reversibility; and the Geometric Origin Displacement Index (GODI), a proposed—not operationalised, not validated—index of deviation from GOB. The electromechanical proposal is stated as three nested hypotheses. H1, the broad electromechanical hypothesis, holds that deformation of collagen-rich matrix generates local electrical phenomena—electrokinetic, piezoelectric and potentially flexoelectric—which may modulate the behaviour of matrix-producing cells. H2, nested within H1, holds that a solid-state component contributes at physiological hydration and, because it does not require fluid flow for its generation, would be expected to persist when electrokinetic contributions are reduced. H3, nested within H2, holds that this solid-state component is specifically piezoelectric rather than flexoelectric. Each may fail without the one above it failing. We further propose that chronic load asymmetry is distributed through mechanically continuous tissue and generates locally biased electrical signalling at many sites, rather than an electrical field propagating through the organism. If cellular remodelling phenotype is unchanged when electromechanically active and electrically inert matrices are compared under matched mechanical exposure, across the substrate, cell type and loading regimes tested, the electrical contribution proposed here would not be supported within that experimental domain.
Electron transfer (ET) is involved in many fundamental biological processes. While spectroscopic and electrochemical techniques have established the theoretical framework governing protein ET, they typically capture average responses across large populations or characterize ET in fixed molecular arrangements. Electrochemical scanning tunneling microscopy (EC-STM) has emerged as a unique single-molecule technique that combines atomic-scale spatial resolution with electrochemical control, enabling direct investigation of charge transport in proteins within a dynamic framework. Initially focused on redox proteins, EC-STM has evolved from imaging to spectroscopic measurements, including current-distance, current-voltage, and break-junction approaches, revealing electrochemically regulated transport pathways and dynamic fluctuations. More recently, EC-STM has been extended to non-redox proteins and to the study of charge transport in increasingly complex biological systems, such as inter-protein ET and molecular recognition. These studies have demonstrated that ligand binding can modulate the electrical response of proteins, providing a direct readout of the conformational and electronic changes associated with biological recognition events. These observations reinforce EC-STM as a powerful approach for studying protein function beyond classical ET systems, bridging the gap between nanoscale biophysics and molecular recognition. This mini review traces the evolution of EC-STM applied to protein ET from its origins in intraprotein ET of redox proteins to its development into a versatile tool for studying protein function through molecular recognition. We analyze how EC-STM has provided new insights into ET mechanisms, protein-protein interactions, and ligand-induced electrical responses, and we conclude by considering its potential for the development of protein-based biosensors and bioelectronic technologies.
Our understanding of protein structure rests on four pillars. Primary sequence is followed by secondary structural motifs. The third pillar is the three-dimensional organisation of single polypeptide chains. The final level includes associations between individual polypeptide chains, and incorporation of structurally essential non-peptide ligands. AlphaFold and other tools can now reliably predict tertiary structure. However, quaternary structure remains relatively poorly understood and is the least predictable element. This is at least in part because quaternary structure is the most contextually governed level. Recent data, including our own work from ribonucleotide reductase and KEAP1, show that a deeper understanding of dynamic associations and factors, such as post-translational modifications, regulating those interactions is necessary to understand and predict quaternary structure. This is particularly true in cells, where we propose that quaternary structure is better conceptualized as a state space encompassing oligomerization, binding partner availability, post-translational modifications, and localization, all governed by signaling context. We discuss how such a conceptualization helps understand and describe how quaternary structure alterations are brought about by signaling changes. Methods to decode these state-gated, structural changes, and implications for gain-of-function drug mechanism and design are also addressed. We put these considerations in context of recent protein-protein interaction prediction models, interactome profiling studies, and functional omics methods that together may be combinable to predict new protein functions linked to dynamic quaternary association changes.
Biomolecular condensates capture the scientific imagination by executing dazzling 3D tours en l’air within the bulk cellular matrix, driven by an intricate theater of multivalent, electrostatic, hydrophobic, and π -driven interactions. However, directing the analytical spotlight onto the 2D planar lipid boundary reveals an exotic, self-regulating pas de deux between fluid condensates and lipid membranes. Operating via a strict attract-repel elastocapillary cycle, highly fluid liquid-disordered domains act as thermodynamic sinks, recruiting condensates and lowering the nucleation energy barrier to drive phase separation. Condensate membrane wetting inevitably dehydrates the underlying interface, triggering a reciprocal phase inversion into a tightly packed, liquid-ordered state that actively repels the condensate and terminates the event. Pathological cellular conditions frequently disrupt this elegant thermodynamic duet; thus, precise stoichiometric calibration and deployment of interfacial linactants represents a transformative therapeutic frontier. Mastering this boundary modulation offers an exceptional opportunity to mechanically choreograph this bidirectional coupling, restoring the neoclassical pas de deux between 3D biomolecular condensates and 2D planar circuitry in health and disease.
