Biology subjects

Perrone, B.

Publications and source records attributed to Perrone, B..

6 recordsLinked to original sources

bioRxiv2026

Integrated solid/solution NMR assignment allows mapping dynamics and ligand binding in a 134 kDa enzyme

Understanding enzyme function requires characterizing not only static structure but also dynamics and ligand interactions. NMR spectroscopy provides this insight at atomic resolution, yet for large proteins the difficulty of resonance assignment has largely confined such studies to systems below[~] 50 kDa, or to observing only methyl groups. Here we present an integrated magic-angle spinning (MAS) and solution NMR study of the 134 kDa tetrameric malate dehydrogenase from Ignicoccus islandicus (IiMDH), an enzyme of particular interest as an evolutionary intermediate between allosteric lactate dehydrogenases and non-allosteric malate dehydrogenases. By combining high-dimensional (up to 4D) MAS NMR experiments on sedimented protein with solution NMR, we achieved 92% backbone heavy-atom assignment and 91% assignment of all Ile-{delta}1, Leu-{delta}1/-{delta}2, Val-{gamma}1/-{gamma}2, Met-{varepsilon} and Thr-{gamma} methyl groups. Building on these assignments, MAS NMR 15N rotating-frame relaxation (R1p) measurements revealed pronounced microsecond-timescale backbone dynamics in functionally critical regions, including the catalytic loop and the mobile surface loop. Complementary methyl-axis order parameters from solution NMR identified additional flexible sites in the hydrophobic core. Chemical shift perturbation experiments upon addition of the substrate analogue oxamate, monitored via backbone 1H-15N TROSY, revealed both active-site contacts and rearrangements of helices a2F and a3G, regions implicated in allosteric signal transmission. The integrated approach demonstrated here exploits the distinct strengths of MAS and solution NMR, and provides a comprehensive view of structure, dynamics, and substrate interactions in a large oligomeric enzyme that would not be accessible by either technique alone.

biophysics↗

bioRxiv2026

Co-sedimentation is the key to the structural investigation of wild-type FAT10

Under inflammatory conditions, the ubiquitin-like modifier FAT10 serves as a tag for protein degradation by the 26S proteasome. FAT10 is degraded along with its substrates and this process is independent of the segregase VCP/p97, which, in the regular ubiquitin pathway of degradation, is required if a substrate lacks a disordered initiation region. FAT10 itself is loosely folded and its tendency to aggregate has complicated investigations of its structure, interaction, and function. Recently hydrogen-deuterium exchange in combination with mass spectrometry has suggested that, in preparation of degradation by the proteasome, the adapter protein NUB1 traps FAT10 in a mostly unfolded state by capturing a {beta}-strand. {beta}-strand capture was subsequently confirmed by magic-angle spinning (MAS) NMR spectroscopy of a stabilized variant of the N-domain of FAT10 in complex with NUB1L, the longer splice variant of NUB1. MAS NMR, in addition, revealed that the N-domain of FAT10 and NUB1L form a fuzzy complex and that the N-terminus of FAT10 is positioned for initiation of degradation by specific non-covalent interaction with NUB1L. Here, we report the investigation of the wild-type N-domain of FAT10 by MAS NMR. Co-sedimentation with NUB1L yields high-quality spectra, which enable sequential assignment of resonances. Through the lens of MAS NMR, the complexes of the wild-type and stabilized N-domain of FAT10 with NUB1L are identical. The N-terminus of FAT10 again shows up prominently in the spectra, even though the residue is this time an Ala, not a Gly. Our experience suggests that co-sedimentation in combination with MAS NMR is generally helpful in the exploration of conditional folds of intrinsically disordered proteins.

biochemistry↗

bioRxiv2025

Calorie restriction leads to degradation of mutant uromodulin and ameliorates inflammation and fibrosis in UMOD-related kidney disease

BackgroundMutations in UMOD, encoding uromodulin, lead to Autosomal Dominant Tubulointerstitial Kidney Disease (ADTKD), a genetic cause of kidney failure. UMOD mutations have a common gain-of-toxic-function effect, causing mutant uromodulin retention in the endoplasmic reticulum (ER). This leads to ER stress, alteration of protein homeostasis and mitochondrial dynamics, defective autophagy and increased cell death. Calorie restriction (CR) exerts a beneficial role in diseases characterized by accumulation of pathogenic protein and inflammation, by modulating several pathways, including autophagy induction and suppression of inflammation and fibrosis. Given the relevance of these features in ADTKD, we investigated the effect of CR on disease onset and progression. MethodsTransgenic mice expressing C147W uromodulin (TgUmodC147W) were subjected to a moderate (30%) CR regimen for 15 or 24 weeks, starting at different stages of disease progression. ResultsCR restored autophagy, as shown by decreased P62 punctae and quenched mTOR activation specifically in mutant uromodulin expressing cells, it recovered expression of key ER-phagy receptor genes, with a concomitant, striking reduction of mutant uromodulin ER retention. In pre-symptomatic TgUmodC147W mice, CR alleviates epithelial cell stress. This, likely along with a direct anti-inflammatory effect of CR, prevents inflammation and progressive decline of kidney function. At this early disease stage, CR ameliorates the already established kidney damage and reduces fibrosis, suggesting reversal of ADTKD phenotype. CR was also effective in significantly delaying disease progression in TgUmodC147W mice with advanced disease and already compromised kidney function. ConclusionOur findings establish the proof-of-principle that counteracting the primary effect of UMOD mutations by stimulating autophagy and quenching inflammation prevents disease onset and progression. This study uncovers the potential of CR as a valuable therapeutic option in the context of ADTKD-UMOD, and possibly other proteinopathies. Key pointsO_LICalorie restriction (CR) stimulates autophagy to degrade mutant uromodulin leading to amelioration of cell stress and tubular damage. C_LIO_LIAt early disease stage CR reverts ADTKD-UMOD phenotype, by preventing inflammation and progressive decline of kidney function and rescuing fibrosis. C_LIO_LIAt advanced disease stage, CR significantly delays disease progression and worsening of kidney function. C_LI

genetics↗

bioRxiv2025

MAS cryoprobe enhances solid-state NMR signals of α-synuclein fibrils

Solid-state NMR spectroscopy is increasingly applied to structural and dynamics studies across a broad range of chemical, material, and biological systems. Although sensitivity has traditionally been a major limitation, the recently developed MAS cryoprobe has been shown to substantially overcome this challenge. Its ability to enhance the signal-to-noise (S/N) ratio without requiring sample freezing makes it particularly attractive for investigating non-isotropic systems, including soft materials (e.g., hydrogels), semi-solids (e.g., membrane mimetics) and rigid solids (e.g., amyloid fibrils). In this study, we report on the enhanced sensitivity of solid-state NMR experiments on -synuclein fibrils using a MAS cryoprobe. Nearly an order-of-magnitude improvement in S/N was observed in CPMAS, refocused-INEPT and 2D 13C-13C chemical shift correlation spectra of -synuclein fibrils compared with data collected on a conventional MAS probe. The improved S/N enables the acquisition of slowly decaying signals in the indirect dimension, facilitating faster, high-resolution multidimensional solid-state NMR spectroscopy. We therefore anticipate that MAS cryoprobe will become increasingly valuable for structural studies a wide range of samples that are less abundant, less stable, or transient, such as amyloid intermediates.

biophysics↗

bioRxiv2023

Production of isotopically enriched high molecular weight hyaluronic acid and characterization by solid-state NMR

Hyaluronic acid (HA) is a naturally occurring polysaccharide that is abundant in the extracellular matrix (ECM) of all vertebrate cells. HA-based hydrogels have attracted great interest for biomedical applications due to their high viscoelasticity and biocompatibility. In both ECM and hydrogel applications, high molecular weight (HMW)-HA can absorb a large amount of water to yield matrices with a high level of structural integrity. To understand the molecular underpinnings of structural and functional properties of HA-containing hydrogels, few techniques are available. Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for such studies, e.g. 13C NMR measurements can reveal the structural and dynamical features of (HMW) HA. However, a major obstacle to 13C NMR is the low natural abundance of 13C, necessitating the generation of HMW-HA that is enriched with 13C isotopes. Here we present a convenient method to obtain 13C- and 15N-enriched HMW-HA in good yield from Streptococcus equi subsp. zooepidemicus. The labeled HMW-HA has been characterized by solution and magic angle spinning (MAS) solid-state NMR spectroscopy, as well as other methods. These results will open new ways to study the structure and dynamics of HMW-HA-based hydrogels, and interactions of HMW-HA with proteins and other ECM components, using advanced NMR techniques.

biophysics↗

bioRxiv2021

Imaging active site chemistry and protonation states: NMR crystallography of the tryptophan synthase α-aminoacrylate intermediate

NMR-assisted crystallography - the synergistic combination of solid-state NMR, X-ray crystallography, and first-principles computational chemistry - holds remarkable promise for mechanistic enzymology: by providing atomic-resolution characterization of stable intermediates in the enzyme active site - including hydrogen atom locations and tautomeric equilibria - it offers insight into structure, dynamics, and function. Here, we make use of this combined approach to characterize the -aminoacrylate intermediate in tryptophan synthase, a defining species for pyridoxal-5'-phosphate-dependent enzymes on the {beta}-elimination and replacement pathway. By uniquely identifying the protonation states of ionizable sites on the cofactor, substrates, and catalytic side chains, as well as the location and orientation of structural waters in the active site, a remarkably clear picture of structure and reactivity emerges. Most incredibly, this intermediate appears to be mere tenths of angstroms away from the preceding transition state in which the {beta}-hydroxyl of the serine substrate is lost. The position and orientation of the structural water immediately adjacent to the substrate {beta}-carbon suggests not only the fate of the hydroxyl group, but also the pathway back to the transition state and the identity of the active site acid-base catalytic residue. Reaction of this intermediate with benzimidazole (BZI), an isostere of the natural substrate, indole, shows BZI bound in the active site and poised for, but unable to initiate, the subsequent bond formation step. When modeled into the BZI position, indole is positioned with C3 in contact with the -aminoacrylate C{beta} and aligned for nucleophilic attack. Significance StatementThe determination of active site protonation states is critical to gaining a full mechanistic understanding of enzymatic transformations; yet hydrogen positions are challenging to extract using the standard tools of structural biology. Here we make use of a joint solid-state NMR, X-ray crystallography, and first-principles computational approach that unlocks the investigation of enzyme catalytic mechanism at this fine level of chemical detail. For tryptophan synthase, this allows us to peer along the reaction coordinates into and out of the -aminoacrylate intermediate. Through this process, we are developing a high-resolution probe for structural biology that is keenly sensitive to proton positions - rivaling that of neutron diffraction, yet able to be applied under conditions of active catalysis to microcrystalline and non-crystalline materials.

biophysics↗