Biophysical Chemistry

Glycation, a non-enzymatic reaction occurring between sugars and biological macromolecules, plays a critical role in ageing and disease pathogenesis. Methylglyoxal (MG) is a highly reactive -oxoaldehyde that leads to the formation of endogenous advanced glycation end products (AGEs). These AGEs are associated with diabetes and many other diseases, including neurodegeneration and cancer. This is often through interactions with the receptor for advanced glycation end products (RAGE). Inhibition of glycation/AGEs formation using natural products to target cancer is an area of recent interest. In vitro AGEs formation was observed by browning of samples, increased fluorescence, and carbonyl stress. MG induced changes in the structure of BSA were analysed using electrophoresis, spectroscopy, TEM, AFM, DLS, and CD spectroscopy. Our results show that AGEs form random structures, oligomeric aggregates, and {beta}-sheets. Thioflavin T and Congo red staining further validated these findings. Galangin and Caffeic acid demonstrated significant antiglycation activity, suppressing AGEs formation in vitro. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/737425v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@113b391org.highwire.dtl.DTLVardef@7208a1org.highwire.dtl.DTLVardef@94c2e1org.highwire.dtl.DTLVardef@867b85_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIMethylglyoxal-induced Advanced Glycation End Products were prepared in vitro C_LIO_LIMethylglyoxal -induced structural modifications in BSA C_LIO_LIAGEs were characterised using various parameters C_LIO_LIBoth fluorescent and non-fluorescent AGEs were formed. C_LIO_LIPhytochemical treatment induced inhibition of AGEs formation C_LI

Imipramine is known as an older generation tricyclic antidepressant drug. It has been identified in prior studies that imipramine blocks Apolipoprotein E4 (ApoE4)-induced amyloid-{beta}(A{beta}) aggregation and is associated with an improved AD diagnosis [Johnson et al. Alzheimers Research Therapy, 2022, 14, 88]. Using NMR methods such as 1H-1H NOESY and Saturation Transfer Difference Spectroscopy, we demonstrate the binding of A{beta} monomers to imipramine when the full-length A{beta} (1-42) sequence is considered. The more abundant but less toxic form, A{beta} (1-40) does not show interaction with imipramine.

This paper presents and critically reviews the results of original and some literature based experimental studies conducted by the authors last years on the structural organization of DNA in dormant (starvation stress), anabiotic dormant (4 HR treatment) E. coli cells, as well as the K12 {Delta}dps strain, which lacks the Dps protein (Dps null E. coli). The experimental data includes small-angle synchrotron radiation diffraction (SAXS) and transmission electron microscopy (TEM) data. Synchrotron radiation diffraction experiments on K12{Delta}dps cells allowed us to conclude that peaks at 44.3, 22.1, and 14.8 angstrom resolutions are associated exclusively with ordered DNA organization. Peaks at 44.3, 22.1, and 14.8 angstrom resolutions are also observed for samples of dormant (starvation stress) cells and anabiotically dormant cells. Therefore, this ordered DNA organization also applies to samples of dormant and anabiotically dormant cells. A model is proposed that considers the ordered DNA organization in the cell as a cholesteric liquid crystal. The powder diffraction pattern calculated based on this model is compared with experimental small angle X ray scattering (SAXS) data obtained on Dps-null cell samples. The model completely reproduces the key features of the experimental diffraction pattern from Dps-null cell samples. Accordingly, the cholesteric liquid crystal model corresponds to DNA packaging in dormant and anabiotically dormant cells. Cholesteric liquid crystal ordering should be further considered in all models of cellular DNA packaging. To address the question of which structural organization of DNA predominates in the cell: the cholesteric liquid crystal or nanocrystalline or whether they coexist and fully manifest themselves under different external conditions, it is necessary to utilize the latest methodological advances in structural analysis.

SARS-CoV, MERS-CoV, and SARS-CoV-2 exemplify the persistent threat posed by coronaviruses, with their capacity for zoonotic spill over, rapid transmission, and high mortality, and thus underscores the urgent need for broad-spectrum antiviral strategies. The nucleocapsid (N) protein, essential for RNA binding, genome packaging, and viral replication, is highly conserved among coronaviruses but remains an underexplored antiviral target. In our earlier work, we identified two small molecules, ceftazidime and sennoside A, that bind the N-terminal domain of the SARS-CoV-2 N protein and inhibit nucleic acid binding, and identified their binding sites using NMR chemical shift perturbation assays. Here, we observed that several residues involved in inhibitor binding are conserved across betacoronaviruses, suggesting a shared druggable vulnerability. We have purified recombinant N proteins from SARS-CoV, MERS-CoV, and SARS-CoV-2, and demonstrated by electrophoretic mobility shift assays that both compounds significantly reduced RNA binding. Their inhibitory concentrations (IC50) were determined using fluorescence polarization. The docking analyses indicated that both inhibitors target the RNA-binding pocket of the N-NTD, consistent with a conserved mechanism of action. Collectively, our findings reveal a conserved RNA-binding vulnerability in coronavirus N proteins and highlights the pan-coronavirus therapeutic potential of these inhibitors.

Neutron scattering is a powerful technique to investigate atomic structures and molecular dynamics of proteins at the nano-scale. When it comes to dynamics, incoherent and coherent scattering respectively provide information on the single and collective dynamics of nuclei. In proteins, hydrogen has the highest incoherent cross-section, and it is common practice to overlook the contribution of coherent terms stemming from all nuclei. However, the fast collective dynamics of heavier nuclei could also be studied if coherent scattering and incoherent scattering were experimentally separated. The recent advent of polarized neutron spectroscopy with sufficient flux and energy resolution has made it possible, and opens new perspectives to investigate the relative importance of coherent scattering and the information it provides on biological samples. The present study reports on the use of polarized quasi-elastic neutron scattering (QENS) and the application of a minimalistic model adapted to both individual and collective dynamics. Using a perdeuterated green fluorescent protein as a model globular protein, the study provides an interpretation of the dynamical parameters obtained with QENS, and a comparative study of the Elastic Coherent and Incoherent Scattering Factor. Based on both experiments and calculations, we discuss the relative importance of distinct and self components of coherent scattering, which is often wrongly assumed to be representative of collective dynamics only. The results highlight the current impediments rendering complicated a straightforward analysis of fast collective dynamics in hydrated protein samples.

Eukaryotic translation initiation is tightly regulated by interactions among translation initiation factors (eIFs) that ensure accurate start codon selection. The translation regulator, eIF5 mimic protein 1 (5MP1) contributes to this process by competing with eIF5 for binding to eIF2, thereby increasing the stringency of translation initiation. Despite its important regulatory role and emerging involvement in tumorigenesis, structural information on human 5MP1 remains limited. Here, we report the near-complete backbone and partial side-chain NMR resonance assignments of the C-terminal domain of human 5MP1 (residues 250-419), carrying a W404E substitution that disrupts dimerization. The WT protein forms a dimer at NMR concentrations, which increases the effective size of the protein and also causes disappearance of peaks corresponding to aminoacids at the dimer interface due to conformational exchange. Backbone resonance assignments were completed for 96.4% of the non-proline residues. Secondary structure was analyzed using Chemical Shift Index (CSI) and compared with the AlphaFold structural model. Regions of disagreement between the experimental and computational secondary structure assignments were further examined using 15N-NOESY-HSQC spectra, allowing experimental validation of local structural features. While the AlphaFold model accurately reproduces the overall fold of the 5MP1 C-terminal domain, several localized discrepancies were identified, particularly near the N- and C-terminal regions of the domain, where experimental NMR data support alternative secondary structure assignments. These resonance assignments and experimentally validated structural features provide a foundation for future investigations of the molecular interactions, dynamics, and functions of 5MP1 in translation initiation.

Streptavidin-biotin, avidin-biotin interactions are classical models for protein-ligand binding, yet the energetic changes accompanying biotin binding remain poorly resolved. Using fluorescent dyes as energy sensors, we show that biotin binding produces two distinct regimes occurring in parallel as concentration of biotin increases cooperativity and conformational rearrangements, wherein cooperativity is observed via exchange broadening of fluorescence linewidth and conformational rearrangements exclusively observed in emission energy. Where the first biotin binding creates the highest contribution to the emission energy. Further analysis of tetramer-tetramer only interactions revealed extremely long ranged intermolecular interactions extending to hundreds of nm. The intermolecular interactions become negligible only at concentrations of approximately 10 nM for both streptavidin and avidin. Affinity values estimated for these diluted samples were below 1 nM.

Amylose contributes to starch crystallinity, but the stability of packed amylose double helices in water at elevated temperature remains insufficiently characterized. Here, we used molecular dynamics simulations to test whether chain length affects the short-timescale stability of A-type amylose oligomers in water. Six systems differing in chain length (6, 12, or 24 glucose units per chain) and oligomer size (isolated double strand or dodecamer of six double strands) were simulated, and five independent 1-s production runs were analyzed for each simulated condition. Oligomers with six glucose units showed structural collapse accompanied by increased water penetration. By contrast, dodecamers with 12 or 24 glucose units largely retained packed double-helical organization over the simulated timescale, although fraying was observed at their ends. These results indicate that chain length and lateral packing strongly affect the early structural response of amylose-like crystalline segments in hot water. The present simulations do not establish the ultimate fate of longer oligomers at longer timescales, but they identify a relative stability difference that is relevant to molecular interpretations of hydration-driven disordering in starch.

Reconstructing time-resolved inter-residue distance distributions during protein functional dynamics in the solution state is known to be a difficult and important problem. This article presents a technique for extracting spin-spin (as a proxy for residue-residue) distance distributions on doubly-spin-labeled proteins from rapid-scan time-resolved Gd-Gd electron paramagnetic resonance (rs-TiGGER) spectra recorded near room temperature in solution at 240 GHz. We use a best-fit technique that convolves a dipolar kernel matrix with an intrinsic, non-dipolar-broadened (single-labeled) spectrum. The kernel incorporates the effect of solution-state tumbling on the dipolar broadening using a correlation function that bridges the static and rapidly tumbling regimes. We apply the technique to AsLOV2, a protein domain with a dark-state crystal structure that is well-known from X-ray crystallography, but a less well-characterized and disordered tertiary structure that manifests after photoactivation at 450 nm. Informed by principal component analysis, we assume that the underlying distance distribution may be approximated by a sum of two Gaussian distributions. The fits returned time-resolved, light-activated populations with mean distances of [Formula] (dark) and [Formula] (lit) in the wild type, and [Formula] (dark) and [Formula] (lit) in an N414Q mutant, with nearly complete unfolding (within fit uncertainty) of the active, light-sensitive fraction. The extracted distance distributions and their accompanying uncertainties are consistent within uncertainty with molecular dynamics simulations of the equilibrated protein structure.

The widespread use of synthetic food dyes, such as Acid Yellow 23 (AY 23), in the food, cosmetics, and pharmaceutical industries raises questions about their potential effects on biological systems and public health. The concentration-dependent interaction between AY 23 and bovine serum albumin (BSA), a crucial model protein for understanding pharmacokinetics and protein-ligand behaviour, was examined in this study. We demonstrate that, under physiological conditions, increasing dye concentrations from 50 M to 200 M results in notable conformational changes, increased surface hydrophobicity, and protein aggregation using a multimodal biophysical approach that includes fluorescence spectroscopy. Direct visualisation verified these structural changes and aggregate formation, whereas hemolytic assay confirmed the high hemolytic nature of AY 23-induced fibrils. Additionally, this study provides a mechanistic basis for the toxicological effects of AY 23, underscoring the implications of food dyes for public health.

Magnetic treatment in tomato seeds (Solanum lycopersicum L.) has been studied as a biotechnological technique to induce a reduction in germination times and enhance plant development. However, the modified cellular mechanisms involved in the reduction of germination times or the improvement of development parameters are not yet clearly established. To explore a possible altered cellular mechanism, the effect of homogeneous static magnetic fields on the structure of the cyclic nucleotide-gated channel 6 (CNGC6), the modification in the organization of POPC lipids in the plasma membrane, and changes in calcium ion mobility were evaluated. For this purpose, coarse-grained molecular dynamics simulations were performed using the Martini 3 model in GROMACS, applying five different magnetic flux densities (0.000, 0.001, 0.010, 0.100, 1.000, and 10.000) T over 1 000 ns. The results showed an anisotropic effect in the longitudinal direction of the protein, which generated heterogeneous behavior among the chains of the homotetramer; this altered the conformation of the CNGC6 channel and modified the pore bottleneck. In contrast, no significant changes were observed in the conformational order of the POPC phospholipid chains. As a preliminary, single-replicate exploratory study, these results suggest that homogeneous static magnetic fields may induce specific structural modifications in the CNGC6 ion channel of Solanum lycopersicum L. without compromising the integrity of the lipid bilayer or the dynamics of ion transport within the analyzed timescale; these preliminary findings provide a molecular-level structural basis for future experimental and computational investigations of magnetic field effects on plant cyclic nucleotide-gated channels.

The aggregation of -synuclein (Syn) is a molecular hallmark of Parkinson's disease (PD) and other synucleinopathies. Understanding the molecular mechanisms that determine the aggregation of this protein may thus facilitate the development of disease-modifying therapies. While Syn is most commonly expressed as a 140-residue protein (Syn-140), recent evidence suggests an involvement of alternatively spliced Syn isoforms in disease onset and progression. Here, we report and characterise the interaction between Syn-140 and the aggregation-prone Syn-112 variant, one of the most abundant Syn splice isoforms. We found that amounts as low as 1% of Syn-112 accelerate the nucleation and aggregation of Syn-140. To further investigate this phenomenon, we employed MALDI-MS and NMR spectroscopy, confirming that Syn-140 and Syn-112 monomers interact strongly with one another. Furthermore, to assess the association of Syn-112 with disease pathology, we performed immunohistochemical staining combined with confocal microscopy on PD brain samples. Thereby, we found an increase in the number as well as the area of Syn-112 immunoreactive aggregates compared to healthy controls. These results illustrate how low-abundance Syn splice isoforms can modulate the aggregation landscape of Syn-140 and in turn contribute to the molecular heterogeneity of synucleinopathies.

We present the solution structures of HIV-1 proteins NC(p7)1-55 corresponding to the full-length NC(p7) and mature p6. The studies were carried in water and, to mimic the membrane, in micellar DPC (Dodecylphosphocholine) conditions. Our results unravel for the first time the structure adopted by the N-terminal amino acids of the free NC(p7)1-55, with the formation of a small helix spanning residues F6 to R10. Our NMR and Fluorescence Anisotropy data disclose an interaction between NC(p7)1-55 and p6 both in water and DPC, with respective Kd of 2.5mM and 370 mM at 23{degrees}C. The interaction is thus strengthened in lipidic conditions. Protein p6 stabilizes the N-terminus of NC(p7)1-55 while increasing at the same time the dynamic of the first zinc finger. Although the entire p6 sequence is involved in the interaction, we show that its C-terminal region is particularly sensitive to the presence of NC(p7)1-55, with a propensity of forming a a helix ranging from amino acids S111 to F116. This study brings experimental evidence of a direct protein-protein interaction between p6 and the N-terminal region of NC(p7)1-55. We further show that such interaction is readily accommodated within the NC(p15) framework and hypothesize that it may facilitate the selective assembly of assembly of the viral genomic RNA (gRNA) in the cell.

Production of recombinant proteins frequently yields inclusion bodies that must undergo refolding to yield active protein. Here, we optimized the refolding conditions for the recombinant leucyl aminopeptidase (rPepL) from Lactocaseibacillus casei expressed in inclusion bodies from E. coli. Several chemical additives were assessed for how well they facilitated an increase in refolding efficiency. The best, 0.5 M L-arginine, yielded 50.8% refolding. The addition of stabilizers, such as sucrose and glycerol, with L-arginine further increased yields to 85%. Urea at lower concentrations (0.25-0.5 M) also facilitated an increase in the refolding yield when co-added with L-arginine, whereas guanidinium chloride inhibited it. Sugars and polyols exhibited dose-dependent effects, with ranges for optima also defined. Fluorescence spectroscopy verified enhancements in the refolding under the optimized conditions. Molecular dynamics simulation under mixed solvent conditions provided atomic insights about stabilizing interactions that are likely to facilitate increased refolding. The results show that a series of aggregation suppressors and protein stabilizers can, in a collaborative way, increase the refolding efficiency for the recombinant proteins from the inclusion bodies. The protocol with the optimization using the additives L-arginine, sucrose, and glycerol is an efficient method for the production of active rPepL. This article outlines the best refolding method to recover recombinant leucyl aminopeptidase from inclusion bodies of E. coli using L-arginine combined with sucrose and glycerol. The combined experimental observations and computational simulations elucidate the molecular process of additive-induced stabilization, which elucidates how aggregation inhibition and hydrogen-bonded stabilization act synergistically. The results presented herein answer both mechanistic understanding and experimental guidance for improving protein refolding.

Schistosoma mansoni is a vector-borne intestinal parasite, endemic in [~]70 tropical countries. Most of the World Health Organization interventions are based on mass drug administration (MDA) and sanitation. Since the parasite does not induce permanent immunity, reinfection rate is high, inducing scheduling of several MDA campaigns. To follow up after treatment and avoid blind MDA, providing a new rapid and cheap diagnostic test is needed, since the most used method is fifty years-old, lacks sensitivity and efficiency. Venom Allergen-Like proteins (SmVALs) have been previously identified as secreted/excreted proteins and potential biomarkers. Here we present the biophysical characterization of SmVAL11 and SmVAL13, their recognition by both specific antibodies and molecularly imprinted polymers (nanoMIPs), an easy-to-standardize alternative. The specific antibodies showed no cross-reactivity, while initial characterization of the nanoMIPs indicates a promising level of selectivity, establishing them as a reliable, cost-effective alternative. Therefore, our results are promising for the future development of a new cheap diagnostic test of intestinal schistosomiasis.

Attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy of proteins in aqueous solution is often limited by water absorption and other optical artifacts. To overcome these limitations, we evaluated the structural features and hydrogen-deuterium exchange (HDX) kinetics of the -helical protein GCN4 in both hydrated (wet) and vacuum-dried (dry) states. While solvent heavily mask the second-derivative spectra of wet samples, vacuum drying yielded a thin, protein-rich film on the ATR crystal, significantly enhancing the signal-to-noise ratio and resolving the protein features without altering the native structure. Dry-state analysis clearly resolved the Amide I, Amide II, and deuterium-shifted Amide II' (1450 cm-1) bands. Notably, second-derivative analysis of the dry spectra of the HDX samples revealed a bimodal Amide I distribution consisting of a stationary band at 1653 cm-1 from the solvent-inaccessible regions and an isotopically sensitive band shifting from 1648 cm-1 to 1644 cm-1 from solvent-accessible regions. These results demonstrate that vacuum-dried ATR-FTIR spectroscopy effectively eliminates solvent masking, providing the spectral clarity required to resolve discrete -helical sub-populations after deuteration.

Botanical dietary supplements (like wheat, barley, teff, oats, white lupin, pumpkin seed, and chickpeas) may contain trace amounts of toxicants in addition to important micronutrients. Developing and validating a reliable protocol for the simultaneous quantification of Cu, Fe, Zn, Mo, Se, Mn, Pb, Al, Ni, and Cr using a PerkinElmer (NexIONTM2000 model) quadrupole ICP MS (including a He collision and reaction cell when needed) with closed vessel microwave digestion using (HNO3 + H2O2) was the aim of this study.The method was subsequently utilized in a sample survey, and the outcomes were evaluated against WHO/JECFA standards. From five study regions, twenty-seven farm-collected botanical powder samples representing seven species were acquired. To create one composite per species, field subsamples were cleaned, air dried, ground, and blended (nine subsamples per botanical: three grabs from each of three farms). HNO3/H2O2 was used to digest aliquots (0.250-0.500gm) in closed microwave containers. Internal standards, multi-point external calibration, procedural blanks, verified reference materials, matrix spikes, and duplicates were all used in ICP MSs multi-element quantitation. Method LODs/LOQs, accuracy (CRM recoveries), and precision (RSD) were calculated.The technique produced low LODs that were suitable for dietary evaluation (typical LOD ranges: Cu, Fe, Zn, Mn, Ni, Cr (0.001-0.01) mg/kg; Mo, Se, Pb, Al (0.002-0.05) mg/kg. For the majority of analytes, within-run RSDs were less than 5%, while CRM recoveries ranged from 88.9 to 110%. The concentrations of essential elements varied greatly (average mg/kg: Fe (280.7{+/-}25.6); Zn (6.0{+/-}0.541); Cu (2.8{+/-}0.269); Mn (398.3{+/-}23.8); {micro}gm/kg: Se (0.061{+/-}0.006); Mo (1.0 {+/-}0.022). Although some composites approached or exceeded conservative intake thresholds for Pb and Al under high consumption scenarios, toxic elements were generally low (mean mg/kg: Pb (0.062{+/-}0.007); Al(185.2{+/-}18.5); Ni(1.6{+/-}0.163); Cr(1.8{+/-}0.171).For the simultaneous nutritional and contaminant profiling of supplements derived from cereals and those not, the validated ICP- MS workflow with microwave HNO3 and H2O2 digestion is suitable. Accurate labeling and consumer safety can be supported by routine screening and supply chain controls.

Nanodiscs and bicelles are widely used as membrane mimetics for structural studies of membrane-associated systems. Studies have reported that their magnetic alignment behavior and phase stability are highly sensitive to composition and temperature. In this study, we systematically investigate the effects of cholesterol on bicelle formation and magnetic alignment in DMPC + 0.2 glycyrrhizic acid (GA) systems using a combined 31P and 14N solid-state NMR experimental and simulation-based approach. Temperature dependent 31P NMR spectra reveal a clear transition from vesicle dominant to aligned bicelles/nanodsics phase, while 1N quadrupolar splitting and lineshape analysis provides quantitative insights into heterogeneous lipid bilayer populations, distinguishing large aligned nanodiscs (B(L)), small nanodiscs (B(S)), and isotropic/random components (B(R)). A strong correlation is observed between the 31P derived bicelle fraction and the 14N B(L) population, confirming that macroscopic alignment in the presence of an external magnetic field directly reflects the growth of large, well-ordered nanodiscs. Cholesterol is found to play a critical dual role by modulating membrane order and curvature. At low cholesterol concentration (0 to 5 mole percent), nanodiscs alignment occurs gradually with increasing temperature, while at higher cholesterol concentration (15 to 25 mole percent), the alignment is delayed and accompanied by broader spectral features, indicating structural heterogeneity. Notably, 10 mole percent cholesterol consistently provides the optimal balance, enabling efficient temperature dependent conversion to aligned bicelles while maintaining high B(L) populations (about 70-80 percentage) and minimal isotropic fractions. In contrast, higher cholesterol maintains significant B(S) and B(R) populations, even at elevated temperature. The 14N quadrupolar coupling (Cq is approximately 8.5 to 9.2 kHz for aligned nanodiscs) remains nearly invariant across compositions, showing that cholesterol does not change local headgroup dynamics but instead redistributes lipid populations. These findings establish a combined 31P and 14N solid -state NMR approach provides a valuable platform for quantitatively correlating membrane structure, dynamics, and alignment, offering practical guidelines for optimizing bicelle systems for high resolution solid-state NMR studies of membrane associated biomolecules.

The Epstein-Barr virus (EBV) is a highly prevalent virus worldwide that is associated with several lymphoid and epithelial malignancies. However, extensive research on EBV integral membrane proteins BILF1, LMP1 and LMP2, has been scarce due to their hydrophobic transmembrane domains. Our study applies the QTY code (glutamine, threonine, tyrosine) to design water-soluble analogs of BILF1, LMP1 and LMP2 with reduced hydrophobicity, where we systematically replaced hydrophobic amino acid residues leucine (L), isoleucine (I), valine (V), and phenylalanine (F) with structurally similar polar residues glutamine (Q), threonine (T), and tyrosine (Y). We retrieved their native sequences from UniProt, identified transmembrane domains using Protter, then performed QTY design through the Protein Solubilizing Server (PSS). We then predicted native and QTY structures using in silico prediction tools AlphaFold3, ColabFold, and Boltz-2. Our analyses demonstrate that despite significant protein sequence replacements in their transmembrane domains (54.15%-61.59%) and increased intrinsic solubility, the QTY analogs exhibited minimal changes in isoelectric point (0.00-0.15 decrease) and molecular weight (0.7-1.2 kDa increase). Additionally, structural superpositions between QTY analogs and native structures using PyMOL yield low RMSD values (0.217[A] -1.202[A]). Our results demonstrate the QTY codes ability to design detergent-free analogs of BILF1, LMP1 and LMP2 with substantially reduced hydrophobicity and aggregation propensity whilst preserving native-like structures. Our results may facilitate protein characterization studies, therapeutic research on EBV, and other protocols that typically require protein solubilization.

Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1), a key regulator of mitophagy, has been linked to the pathogenesis of Parkinson's disease (PD). PINK1 recruits Parkin, an E3 ubiquitin ligase, triggering mitophagy in response to mitochondrial damage. During mitophagy, the quantity, stability, and activity of PINK1 must be strictly regulated; however, the mechanisms governing these parameters under cellular stress are still unclear. Herein, we determined the structural basis for PINK1 maturation mediated by heat shock protein 90/cell division cycle 37/FK506-binding protein 51 (HSP90/CDC37/FKBP51) chaperone complex. We identified PINK1-associated proteins using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and determined the structures of the complexes using Cryo-Electron Microscopy (Cryo-EM). Results showed that FKBP51 potentially interacts with a conserved leucine-proline-phenylalanine (LPF) motif on the activation loop of PINK1 and negatively regulates PINK1 functions in mitophagy. A PINK1 mutation located at the FKBP51 recognition site is linked to mitophagy deficiency, which can be partially rescued by specific inhibition of FKBP51. These findings reveal a general mechanism for PINK1 recognition by the HSP90/CDC37/FKBP51 chaperone complex and suggest a potential approach for upregulating PINK1 activity, which is impaired in PD.