Publications

Interactions between proteins and metal cations are central to biochemical processes and shape protein structures. SilE, an intrinsically disordered protein involved in bacterial silver-resistance, folds into α-helices upon binding Ag⁺ ions. Focusing on the B1 peptide fragment from SilE, we investigate the mechanism of Ag⁺-induced folding with simulations and NMR experiments. We first derive force-field parameters for Ag⁺-protein interactions using DFT. Then, we use replica-exchange simulations, deep learning and NMR to map B1’s folding landscape and reveal how Ag⁺ shapes it. Specifically, Ag⁺ binding promotes folding by entropic penalizing the disordered state and electrostatic stabilization of the folded state. We also describe how Ag+ alters the folding pathway. Overall, we improve the understanding of metal-induced protein folding and lay the groundwork for further computational investigations of the bacterial silver-resistance machinery.

The emergence of very high NMR magnetic fields will certainly encourage the study of larger biological systems with their dynamics and interactions. NMR spin relaxation allows probing the dynamical properties of proteins where the ¹⁵N longitudinal (R₁) and transverse (R₂) relaxation rates in addition to the ¹H–¹⁵N heteronuclear NOE describe the ps–ns timescale. Their analytical representation involves the chemical shift anisotropy (CSA) effect that represents the major contribution at a very high magnetic field above 18.8 T. An accurate analysis of the latter parameters in terms of model free (MF) requires considering its effect. Until now, a uniform value of −160 ppm for the CSA has been widely used to derive the backbone order parameters (S²), giving rise to a large fluctuation of its value at very high magnetic fields. Conversely, the use of a site-specific CSA improves the accurate analysis of protein dynamics but requires a cost-effective experimental multi-field approach. In the present paper, we show how the CSA mainly contributes to the relaxation parameters at 28.2 T compared to lower magnetic fields and may bias the determination of S². We propose to replace the time-consuming measurement of spin relaxation at multiple fields by a combination of molecular dynamics (MD) and the measurement of spin relaxation at one very high magnetic field only. We applied this strategy to three well-folded proteins (ubiquitin, GB3 and ribonuclease H) to show that the determined order parameters are in good agreement with the ones obtained by means of experimental data only.

A common separation approach for polar compounds involves coupling reversed-phase liquid chromatography (RPLC) with hydrophilic interaction chromatography (HILIC) in two-dimensional chromatography. The higher proportion of acetonitrile used in the HILIC mobile phase, which enhances mass spectrometry detection, encourages its use in the second dimension. Previous studies demonstrated that the HILIC column can be partially equilibrated within very short timeframes without compromising retention time stability, rendering it suitable in online comprehensive two-dimensional liquid chromatography (LC×LC) setups. In addition, a specific number of conditioning cycles seems necessary to establish stable retention times. Here, the repeatability of HILIC when employed as second dimension in LC×LC was investigated, with a focus on determining the required number of conditioning cycles to achieve repeatable retention times. Various parameters influenced by the LC×LC online modulation system were studied, such as steep gradient slopes up to 8%, and very short equilibration times, less than or equal to dead time, as well as injection volume and solvent, which depend on the first dimension. Finally, the use of HILIC as a second dimension with tailored conditioning runs was applied to the analysis of hyaluronic acid hydrogel digests. The application of an RPLC×HILIC method using five conditioning runs yielded exceptional stability in second-dimension retention times.

Conventional therapies for inflammatory bowel diseases are mainly based on systemic treatments which cause side effects and toxicity over long-term administration. Nanoparticles appear as a valid alternative to allow a preferential accumulation in inflamed tissues following oral administration while reducing systemic drug exposure. To increase their residence time in the inflamed intestine, the nanoparticles are here associated with a hydrogel matrix. A bioadhesive peptide-based hydrogel is mixed with nanoemulsions, creating a hybrid lipid-polymer nanocomposite. Mucopenetrating nanoemulsions of 100 nm are embedded in a scaffold constituted of the self-assembling peptide hydrogel product PuraStat. The nanocomposite is fully characterized to study the impact of lipid particles in the hydrogel structure. Rheological measurements and circular dichroism analyses are performed to investigate the system's microstructure and physical properties. Biodistribution studies demonstrate that the nanocomposite acts as a depot in the stomach and facilitates the slow release of the nanoemulsions in the intestine. Efficacy studies upon oral administration of the drug-loaded system show the improvement of the disease score in a mouse model of intestinal inflammation.

Lactate accumulation and acidification in tumours are a cancer hallmark associated with the Warburg effect. Lactic acidosis correlates with cancer malignancy, and the benefit it offers to tumours has been the subject of numerous hypotheses. Strikingly, lactic acidosis enhances cancer cell survival to environmental glucose depletion by repressing high-rate glycolysis and lactic fermentation, and promoting an oxidative metabolism involving reactivated respiration. We used real-time NMR to evaluate how cytosolic lactate accumulation up to 40 mM and acidification up to pH 6.5 individually impact glucose consumption, lactate production and pyruvate evolution in isolated cytosols. We used a reductive cell-free system (CFS) to specifically study cytosolic metabolism independently of other Warburg-regulatory mechanisms found in the cell. We assessed the impact of lactate and acidification on the Warburg metabolism of cancer cytosols, and whether this effect extended to different cytosolic phenotypes of lactic fermentation and cancer. We observed that moderate acidification, independently of lactate concentration, drastically reduces the glucose consumption rate and halts lactate production in different lactic fermentation phenotypes. In parallel, for Warburg-type CFS lactate supplementation induces pyruvate accumulation at control pH, and can maintain a higher cytosolic pyruvate pool at low pH. Altogether, we demonstrate that intracellular acidification accounts for the direct repression of lactic fermentation by the Warburg-associated lactic acidosis.

Interleukin-10 (IL-10) is an anti-inflammatory cytokine that is secreted in response to an acute phase inflammation in patients who are suffering from heart failure (HF). The aim of this work was to develop an electrochemical biosensor for determining salivary IL-10 levels. Biofunctionalization strategy was improved through the use of copper-free click chemistry for the developed sensor due to its advantages, leading to high quantitative yields of stable triazoles, rapid reaction, no cytotoxic Cu(I) catalyst requirement, and high specificity of cyclooctynes toward azides. The approach involved in binding of dibenzocyclooctyne acid (DBCO-COOH) to thiol-azide assembled gold microelectrodes, later capturing the monoclonal IL-10 antibody (IL-10 mAb), and ultimately allowing direct detection of IL-10 antigen. Fourier transform infrared spectroscopy (FTIR) and nanoplotter associated with fluorescence microscopy methods have been employed to analyze and prove the biofunctionalization of the gold microelectrodes. Moreover, the electrochemical impedance spectroscopy (EIS) technique was used for detecting IL-10 antigen. The developed immunosensor showed a semi-logarithmic linear range, from 0.1 pg/mL to 5 pg/mL with R2 = 0.9815 and a limit of quantitation (LOQ) of 0.1 pg/mL with relative standard deviation (RSD) of 10.67%. The specificity of the immunosensor was evaluated using an inflammatory cytokine, and none of it generated detectable EIS signals. Finally, the successful analysis of saliva samples from a healthy volunteer without Coronavirus (COVID-19) infection demonstrated the usefulness of the developed immunosensor.

Genome sequencing of the human parasite Schistosoma mansoni revealed an interesting gene superfamily, called micro-exon gene (meg), that encodes secreted MEG proteins. The genes are composed of short exons (3–81 base pairs) regularly interspersed with long introns (up to 5 kbp). This article recollects 35 S. mansoni specific meg genes that are distributed over 7 autosomes and one pair of sex chromosomes and that code for at least 87 verified MEG proteins. We used various bioinformatics tools to produce an optimal alignment and propose a phylogenetic analysis. This work highlighted intriguing conserved patterns/motifs in the sequences of the highly variable MEG proteins. Based on the analyses, we were able to classify the verified MEG proteins into two subfamilies and to hypothesize their duplication and colonization of all the chromosomes. Together with motif identification, we also proposed to revisit MEGs’ common names and annotation in order to avoid duplication, to help the reproducibility of research results and to avoid possible misunderstandings.

In this paper, a microconductometric sensor has been designed, based on a chitosan composite including alcohol dehydrogenase—and its cofactor—and gold nanoparticles, and was calibrated by differential measurements in the headspace of aqueous solutions of ethanol. The role of gold nanoparticles (GNPs) was crucial in improving the analytical performance of the ethanol sensor in terms of response time, sensitivity, selectivity, and reproducibility. The response time was reduced to 10 s, compared to 21 s without GNPs. The sensitivity was 416 µS/cm (v/v%)⁻¹ which is 11.3 times higher than without GNPs. The selectivity factor versus methanol was 8.3, three times higher than without GNPs. The relative standard deviation (RSD) obtained with the same sensor was 2%, whereas it was found to be 12% without GNPs. When the air from the operator’s mouth was analyzed just after rinsing with an antiseptic mouthwash, the ethanol content was very high (3.5 v/v%). The background level was reached only after rinsing with water.

Micro-Exon Genes are a widespread class of genes known for their high variability, widespread in the genome of parasitic trematodes such as Schistosoma mansoni. In this study, we present a strategy that allowed us to solve the structures of three alternatively spliced isoforms from the Schistoma mansoni MEG 2.1 family for the first time. All isoforms are hydrophobic, intrinsically disordered, and recalcitrant to be expressed in high yield in heterologous hosts. We resorted to the chemical synthesis of shorter pieces, before reconstructing the entire sequence. Here, we show that isoform 1 partially folds in a-helix in the presence of trifluoroethanol while isoform 2 features two rigid elbows, that maintain the peptide as disordered, preventing any structuring. Finally, isoform 3 is dominated by the signal peptide, which folds into a-helix. We demonstrated that combining biophysical techniques, like circular dichroism and nuclear magnetic resonance at natural abundance, with in silico molecular dynamics simulation for isoform 1 only, was the key to solve the structure of MEG 2.1. Our results provide a crucial piece to the puzzle of this elusive and highly variable class of proteins.

The resistance of gram-negative bacteria to silver ions is mediated by a silver efflux pump, which mainly relies on a tripartite efflux complex SilCBA, a metallochaperone SilF and an intrinsically disordered protein SilE. However, the precise mechanism by which silver ions are extruded from the cell and the different roles of SilB, SilF, and SilE remain poorly understood. To address these questions, we employed nuclear magnetic resonance and mass spectrometry to investigate the interplay between these proteins. We first solved the solution structures of SilF in its free and Ag⁺-bound forms, and we demonstrated that SilB exhibits two silver binding sites in its N and C termini. Conversely to the homologous Cus system, we determined that SilF and SilB interact without the presence of silver ions and that the rate of silver dissociation is eight times faster when SilF is bound to SilB, indicating the formation of a SilF–Ag–SilB intermediate complex. Finally, we have shown that SilE does not bind to either SilF or SilB, regardless of the presence or absence of silver ions, further corroborating that it merely acts as a regulator that prevents the cell from being overloaded with silver. Collectively, we have provided further insights into protein interactions within the sil system that contribute to bacterial resistance to silver ions.

Phosphodiesterases (PDEs) are a superfamily of evolutionarily conserved cyclic nucleotide (cAMP/cGMP)-hydrolyzing enzymes, components of transduction pathways regulating crucial aspects of cell life. Within this family, the cGMP-dependent PDE5 is the major hydrolyzing enzyme in many mammalian tissues, where it regulates a number of cellular and tissular processes. Using Kluyveromyces lactis as a model organism, the murine PDE5A1, A2 and A3 isoforms were successfully expressed and studied, evidencing, for the first time, a distinct role of each isoform in the control, modulation and maintenance of the cellular redox metabolism. Moreover, we demonstrated that the short N-terminal peptide is responsible for the tetrameric assembly of MmPDE5A1 and for the mitochondrial localization of MmPDE5A2. We also analyzed MmPDE5A1, A2 and A3 using small-angle X-ray scattering (SAXS), transmission electron microscopy (TEM), structural mass spectrometry (MS) and polyacrylamide gel electrophoresis in their native conditions (native-PAGE) and in the presence of redox agents. These analyses pointed towards the role of a few specific cysteines in the isoforms’ oligomeric assembly and the loss of enzymatic activity when modified.

Silver has been used for its antimicrobial properties to fight infection for thousands of years. Unfortunately, some Gram-negative bacteria have developed silver resistance causing the death of patients in a burn unit. The genes responsible for silver resistance have been designated as the sil operon. Among the proteins of the sil operon, SilE has been shown to play a key role in bacterial silver resistance. Based on the limited information available, it has been depicted as an intrinsically disordered protein that folds into helices upon silver ion binding. Herein, this work demonstrates that SilE is composed of 4 clearly identified helical segments in the presence of several silver ions. The combination of analytical and biophysical techniques (NMR spectroscopy, CD, SAXS, HRMS, CE-ICP-MS, and IM-MS) reveals that SilE harbors four strong silver binding sites among the eight sites available. We have also further evidenced that SilE does not adopt a globular structure but rather samples a large conformational space from elongated to more compact structures. This particular structural organization facilitates silver binding through much higher accessibility of the involved His and Met residues. These valuable results will advance our current understanding of the role of SilE in the silver efflux pump complex mechanism and will help in the future rational design of inhibitors to fight bacterial silver resistance.

A conductometric microsensor for measuring methanol vapor was conceived, based on electrospun composite nanofibers of polyvinyl chloride (PVC) doped with nickel phthalocyanine (NiPc) deposited on interdigitated electrodes (IDEs) as transducers. The nanofiber’s shape, structure, percent atomic content and thermal properties were studied. The methanol sensor showed good sensitivity (505µS.cm⁻¹.(v/v)⁻¹), low limit of detection (LOD = 15 ppm), short response time (13 s), and short recovery time (25 s). The sensor was 4 times more sensitive to methanol than to ethanol and 19 times more sensitive to methanol than to acetone. Furthermore, the sensor response was unaffected by the interfering water vapor, making it more suitable for volatile organic compound (VOC) sensing in the presence of humidity. The sensor was applied for conductometric detection of methanol in rubbing alcohol.

3′-5′ cyclic nucleotide phosphodiesterases (PDEs) are a family of evolutionarily conserved cAMP and/or cGMP hydrolyzing enzymes, components of transduction pathways regulating crucial aspects of cell life. Among them, cGMP-specific PDE5—being a regulator of vascular smooth muscle contraction—is the molecular target of several drugs used to treat erectile dysfunction and pulmonary hypertension. Production of full-length murine PDE5A isoforms in the milk-yeast Kluyveromyces lactis showed that the quaternary assembly of MmPDE5A1 is a mixture of dimers and tetramers, while MmPDE5A2 and MmPDE5A3 only assembled as dimers. We showed that the N-terminal peptide is responsible for the tetramer assembly of MmPDE5A1, while that of the MmPDE5A2 is responsible for its mitochondrial localization. Overexpression of the three isoforms alters at different levels the cAMP/cGMP equilibrium as well as the NAD(P)+/NAD(P)H balance and induces a metabolic switch from oxidative to fermentative. In particular, the mitochondrial localization of MmPDE5A2 unveiled the existence of a cAMP-cGMP signaling cascade in this organelle, for which we propose a metabolic model that could explain the role of PDE5 in some cardiomyopathies and some of the side effects of its inhibitors.

Phenolic and substituted phenol based resoles are commonly used in the formulation of can coatings. However, migration analyses of these coatings are very little described compared to other coating technologies. While epoxy and polyester have well known migrants with defined formation mechanisms, Non-Intentionally Added Substances (NIAS) specifically related to the phenolic resin are hardly studied in the literature. The goal of the publication is to further explore the influence of the phenolic resole, used in the formulation of can coatings, on extracted NIAS's nature. Six different model polyester-phenolic can coatings were formulated each with a specific phenol, cresol or tertbutylphenol-based resole. Can coating films were extracted for 24 h at 40 °C in acetonitrile before analysis. NIAS identification was done using gas chromatography separation coupled to high resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR) spectroscopy analyses. Cyclic polyester oligomers were found in all extracts, with oligomers found in a range of 10 μg/dm² to 226 μg/dm², without specific influence of the resole used in formulation. While very few or no peaks were detected from cresol- and phenol-based resoles, 48 peaks were specifically observed in coating extracts of formulas with tertbutylphenol-based resoles as well as in their respective resoles. The most intense peaks were identified as aldehyde compounds by HRMS and NMR analysis. These aldehydes were semi-quantified in similar proportions as polyester oligomers. The presence of such aldehydes has never been reported in the literature regarding NIAS in can coatings. Further study will then be needed to better understand the aldehyde formation mechanism and assess the toxicological profile of such chemicals.

Syndecans are membrane proteoglycans regulating extracellular matrix assembly, cell adhesion and signaling. Their ectodomains can be shed from the cell surface, and act as paracrine and autocrine effectors or as competitors of full-length syndecans. We report the first biophysical characterization of the recombinant ectodomains of the four human syndecans using biophysical techniques, and show that they behave like flexible random-coil intrinsically disordered proteins, and adopt several conformation ensembles in solution. We have characterized their conformational landscapes using native mass spectrometry (MS) and ion-mobility MS, and demonstrated that the syndecan ectodomains explore the majority of their conformational landscape, from minor compact, globular-like, conformations to extended ones. We also report that the ectodomain of syndecan-4, corresponding to a natural isoform, is able to dimerize via a disulfide bond. We have generated a three-dimensional model of the C-terminus of this dimer, which supports the dimerization via a disulfide bond. Furthermore, we have mapped the NXIP adhesion motif of syndecans and their sequences involved in the formation of ternary complexes with integrins and growth factor receptors on the major conformations of their ectodomains, and shown that these sequences are not accessible in all the conformations, suggesting that only some of them are biologically active. Lastly, although the syndecan ectodomains have a far lower number of amino acid residues than their membrane partners, their intrinsic disorder and flexibility allow them to adopt extended conformations, which have roughly the same size as the cell surface receptors (e.g., integrins and growth factor receptors) they bind to.

NLRP3 controls the secretion of inflammatory cytokines IL-1β/18 and pyroptosis by assembling the inflammasome. Upon coordinated priming and activation stimuli, NLRP3 recruits NEK7 within hetero-oligomers that nucleate ASC and caspase-1 filaments, but the apical molecular mechanisms underlying inflammasome assembly remain elusive. Here we show that NEK7 recruitment to NLRP3 is controlled by the phosphorylation status of NLRP3 S803 located within the interaction surface, in which NLRP3 S803 is phosphorylated upon priming and later dephosphorylated upon activation. Phosphomimetic substitutions of S803 abolish NEK7 recruitment and inflammasome activity in macrophages in vitro and in vivo. In addition, NLRP3-NEK7 binding is also essential for NLRP3 deubiquitination by BRCC3 and subsequently inflammasome assembly, with NLRP3 phosphomimetic mutants showing enhanced ubiquitination and degradation than wildtype NLRP3. Finally, we identify CSNK1A1 as the kinase targeting NLRP3 S803. Our findings thus reveal NLRP3 S803 phosphorylation status as a druggable apical molecular mechanism controlling inflammasome assembly.

SilE and SilB are both proteins involved in the silver efflux pump found in Gram-negative bacteria such as S. typhimurium. Using model peptides along with NMR and CD experiments, we show how SilE may store silver ions prior to delivery and we hypothesize for the first time the interplay between SilB and SilE.

Among the various biophysical methods available to investigate protein dynamics, NMR presents the ability to scrutinize protein motions on a broad range of time scales. ¹H–¹⁵N NMR spin relaxation experiments can reveal the extent of protein motions across the picosecond–nanosecond dynamics probed by the fundamental parameters ¹⁵N-R₁, ¹⁵N-R₂, and ¹H–¹⁵N NOE that can be well sampled by molecular dynamics (MD) simulations. An accurate prediction of these parameters is subjected to a proper description of the rotational diffusion and anisotropy. Indeed, a strong rotational anisotropy has a profound effect on the various relaxation parameters and could be mistaken for conformational exchange. Although the principle of NMR spin relaxation predictions from MD is now well established, numerous NMR/MD comparisons have hitherto focused on proteins that show low to moderate anisotropy and make use of a scaling factor to remove artifacts arising from water model-dependence of the rotational diffusion. In the present work, we have used NMR to characterize the rotational diffusion of the α-helical STAM2-UIM domain by measuring the ¹⁵N-R₁, ¹⁵N-R₂, and ¹H–¹⁵N NOE relaxation parameters. We therefore highlight the use of the polarizable AMOEBA force field (FF) and show that it improves the prediction of the rotational diffusion in the particular case of strong rotational anisotropy, which in turn enhances the prediction of the ¹⁵N-R₁, ¹⁵N-R₂, and ¹H–¹⁵N NOE relaxation parameters without the requirement of a scaling factor. Our findings suggest that the use of polarizable FFs could potentially enrich our understanding of protein dynamics in situations where charge distribution or protein shape is remodeled over time like in the case of multidomain proteins or intrinsically disordered proteins.

Multidomain proteins represent a broad spectrum of the protein landscape and are involved in various interactions. They could be considered as modular building blocks assembled in distinct fashion and connected by linkers of varying lengths and sequences. Due to their intrinsic flexibility, these linkers provide proteins a subtle way to modulate interactions and explore a wide range of conformational space. In the present study, we are seeking to understand the effect of the flexibility and dynamics of the linker involved in the STAM2 UIM-SH3 dual domain protein with respect to molecular recognition. We have engineered several constructs of UIM-SH3 with different length linkers or domain deletion. By means of SAXS and NMR experiments, we have shown that the modification of the linker modifies the flexibility and the dynamics of UIM-SH3. Indeed, the global tumbling of both the UIM and SH3 domain is different but not independent from each other while the length of the linker has an impact on the ps-ns time scale dynamics of the respective domains. Finally, the modification of the flexibility and dynamics of the linker has a drastic effect on the interaction of UIM-SH3 with Lys63-linked diubiquitin with a roughly eight-time weaker dissociation constant.

The polymerase of negative-stranded RNA viruses consists of the large protein (L) and the phosphoprotein (P), the latter serving both as a chaperon and a cofactor for L. We mapped within measles virus (MeV) P the regions responsible for binding and stabilizing L and showed that the coiled-coil multimerization domain (MD) of P is required for gene expression. MeV MD is kinked as a result of the presence of a stammer. Both restoration of the heptad regularity and displacement of the stammer strongly decrease or abrogate activity in a minigenome assay. By contrast, P activity is rather tolerant of substitutions within the stammer. Single substitutions at the “a” or “d” hydrophobic anchor positions with residues of variable hydrophobicity revealed that P functionality requires a narrow range of cohesiveness of its MD. Results collectively indicate that, beyond merely ensuring P oligomerization, the MD finely tunes viral gene expression through its cohesiveness.

A new model of the Zn-based complex, [Zn(LH)(Erx)(ErxH)]ClO₄ (sulfadiazine: LH and enrofloxacin ErxH), has been synthesised with two different, but complementary antibiotics (sulfonamide and quinolon) following an easy procedure. To evaluate the synergetic effect of this multicomponent molecule, the mononuclear complexes [Zn(L)₂(H₂O)(NH₃)] and [Zn(Erx)₃] which each include only one of the two antibiotics (sulfadiazine: LH or enrofloxacin ErxH) were synthesized. All three compounds were characterized, including crystal structure determination by single-crystal X-ray diffraction. For all of the complexes, enrofloxacin is coordinated to Zn(II) by pyridinone and one oxygen atom of the carboxylate group in a monodente mode. The antibacterial activity, determined by the minimum inhibitory concentration on specific bacteria, has been studied on free ligands, on the corresponding mononuclear complexes and on our model.

The SilE protein is suspected to have a prominent role in Ag⁺ detoxification of silver resistant bacteria. Using model peptides, we elucidated both qualitative and quantitative aspects of the Ag⁺-induced a-helical structuring role of His- and Met-rich sequences of SilE, improving our understanding of its function within the Sil system.

¹H−¹⁵N NMR spin relaxation and relaxation dispersion experiments can reveal the time scale and extent of protein motions across the ps−ms range, where the ps−ns dynamics revealed by fundamental quantities R₁, R₂, and heteronuclear NOE can be wellsampled by molecular dynamics simulations (MD). Although the principles of relaxation prediction from simulations are well-established, numerous NMR−MD comparisons have hitherto focused upon the aspect of order parameters S² due to common artifacts in the prediction of transient dynamics. We therefore summarize here all necessary components and highlight existing and proposed solutions, such as the inclusion of quantum mechanical zero-point vibrational corrections and separate MD convergence of global and local motions in coarse-grained and all-atom force fields, respectively. For the accuracy of the MD prediction to be tested, two model proteins GB3 and Ubiquitin are used to validate five atomistic force fields against published NMR data supplemented by the coarse-grained force field MARTINI+EN. In Amber and CHARMM-type force fields, quantitative agreement was achieved for structured elements with minimum adjustment of global parameters. Deviations from experiment occur in flexible loops and termini, indicating differences in both the extent and time scale of backbone motions. The lack of systematic patterns and water model dependence suggests that modeling of the local environment limits prediction accuracy. Nevertheless, qualitative accuracy in a 2 μs CHARMM36m Stam2 VHS domain simulation demonstrates the potential of MD-based interpretation in combination with NMR-measured dynamics, increasing the utility of spin relaxation in integrative structural biology.

The discovery of ubistatins, small molecules that impair proteasomal degradation of proteins by directly binding to polyubiquitin, makes ubiquitin itself a potential therapeutic target. Although ubistatins have the potential for drug development and clinical applications, the lack of structural details of ubiquitin-ubistatin interactions has impeded their development. Here, we characterized a panel of new ubistatin derivatives using functional and binding assays. The structures of ubiquitin complexes with ubistatin B and hemi-ubistatin revealed direct interactions with ubiquitin's hydrophobic surface patch and the basic/polar residues surrounding it. Ubistatin B binds ubiquitin and diubiquitin tighter than a high-affinity ubiquitin receptor and shows strong preference for K48 linkages over K11 and K63. Furthermore, ubistatin B shields ubiquitin conjugates from disassembly by a range of deubiquitinases and by the 26S proteasome. Finally, ubistatin B penetrates cancer cells and alters the cellular ubiquitin landscape. These findings highlight versatile properties of ubistatins and have implications for their future development and use in targeting ubiquitin-signaling pathways.

Using model peptides, each of the nine MX₂H or HX_nM (n = 1, 2) motifs of the silver resistance protein SilE has been shown to coordinate to one Ag⁺ ion by its histidine and methionine residues with K_d in the μM range. This suggests an Ag⁺ buffering role for SilE in the case of high Ag⁺ overload.

Rotational diffusion (D_rot) is a fundamental property of biomolecules that contains information about molecular dimensions and solute–solvent interactions. While ab initio D_rot prediction can be achieved by explicit all-atom molecular dynamics simulations, this is hindered by both computational expense and limitations in water models. We propose coarse-grained force fields as a complementary solution, and show that the MARTINI force field with elastic networks is sufficient to compute D_rot in >10 proteins spanning 5–157 kDa. We also adopt a quaternion-based approach that computes D_rot orientation directly from autocorrelations of best-fit rotations as used in, e.g., RMSD algorithms. Over 2 μs trajectories, isotropic MARTINI+EN tumbling replicates experimental values to within 10–20%, with convergence analyses suggesting a minimum sampling of >50 × τ_theor to achieve sufficient precision. Transient fluctuations in anisotropic tumbling cause decreased precision in predictions of axisymmetric anisotropy and rhombicity, the latter of which cannot be precisely evaluated within 2000 × τ_theor for GB3. Thus, we encourage reporting of axial decompositions D_x, D_y, D_z to ease comparability between experiment and simulation. Where protein disorder is absent, we observe close replication of MARTINI+EN Drot orientations versus CHARMM22*/TIP3p and experimental data. This work anticipates the ab initio prediction of NMR-relaxation by combining coarse-grained global motions with all-atom local motions.