Conformational Changes Research Articles

Exploring Cimetidine as a Potential Therapeutic Attenuator against Amyloid Formation in Parkinson's Disease: Spectroscopic and Microscopic Insights into Alpha-Synuclein and Human Insulin.

Neurodegenerative diseases, notably Alzheimer's and Parkinson's, hallmark their progression through the formation of amyloid aggregates resulting from misfolding. While current therapeutics alleviate symptoms, they do not impede disease onset. In this context, repurposing existing drugs stands as a viable therapeutic strategy. Our study determines the antihistamine drug Cimetidine's potential as an inhibitor using diverse spectroscopic and microscopic methods on alpha-synuclein and human insulin amyloid formation, unveiling its efficacy. The thioflavin T (ThT) assay illustrated a dose-dependent reduction in amyloid formation with escalating concentrations of Cimetidine. Notably, the antihistamine drug maintained a helical structure and showed no significant conformational changes in the secondary structure. Confocal microscopy validated fewer fibrils in the Cimetidine-treated samples. Remarkably, Cimetidine interacted with pre-existing fibrils, leading to their disintegration. Further analyses (ThT, circular dichroism, and dynamic light scattering) showcased the conversion of fibrils into smaller aggregates upon Cimetidine addition. These findings signify the potential of this antihistamine drug as a plausible therapeutic option for Parkinson's disease. This study may open avenues for deeper investigations and possible therapeutic developments, emphasizing Cimetidine's promising role in mitigating neurodegenerative diseases like Parkinson's.

A Demethylation-Switchable Aptamer Design Enables Lag-Free Monitoring of m6A Demethylase FTO with Energy Self-Sufficient and Structurally Integrated Features.

Cellular context profiling of modification effector proteins is critical for an in-depth understanding of their biological roles in RNA N6-methyladenosine (m6A) modification regulation and function. However, challenges still remain due to the high context complexities, which call for a versatile toolbox for accurate live-cell monitoring of effectors. Here, we propose a demethylation-switchable aptamer sensor engineered with a site-specific m6A (DSA-m6A) for lag-free monitoring of the m6A demethylase FTO activity in living cells. As a proof of concept, a DNA aptamer against adenosine triphosphate (ATP) is selected to construct the DSA-m6A model, as the "universal energy currency" role of ATP could guarantee the equally fast and spontaneous conformation change of DSA-m6A sensor upon demethylation and ATP binding in living organisms, thus enabling sensitive monitoring of FTO activity with neither time delay nor recourse to extra supply of substances. This ATP-driven DSA-m6A design facilitates biomedical research, including live-cell imaging, inhibitor screening, single-cell tracking of dynamic FTO nuclear translocation upon starvation stimuli, FTO characterization in a biomimetic heterotypic three-dimensional (3D) multicellular spheroid model, as well as the first report on the in vivo imaging of FTO activity. This strategy provides a simple yet versatile toolbox for clinical diagnosis, drug discovery, therapeutic evaluation, and biological study of RNA demethylation.

3D variability analysis reveals a hidden conformational change controlling ammonia transport in human asparagine synthetase

Advances in X-ray crystallography and cryogenic electron microscopy (cryo-EM) offer the promise of elucidating functionally relevant conformational changes that are not easily studied by other biophysical methods. Here we show that 3D variability analysis (3DVA) of the cryo-EM map for wild-type (WT) human asparagine synthetase (ASNS) identifies a functional role for the Arg-142 side chain and test this hypothesis experimentally by characterizing the R142I variant in which Arg-142 is replaced by isoleucine. Support for Arg-142 playing a role in the intramolecular translocation of ammonia between the active site of the enzyme is provided by the glutamine-dependent synthetase activity of the R142 variant relative to WT ASNS, and MD simulations provide a possible molecular mechanism for these findings. Combining 3DVA with MD simulations is a generally applicable approach to generate testable hypotheses of how conformational changes in buried side chains might regulate function in enzymes.

Affinity purification of the outer hair cell motor protein prestin using His-tag

ObjectiveThe high sensitivity and broad frequency selectivity of mammalian hearing are associated with the somatic motility of outer hair cells (OHCs) in the cochlea. This motility is considered to be induced by conformational changes of the motor protein prestin expressing in the lateral plasma membrane of OHCs. Since its identification in 2000, prestin has been actively investigated and its structure and function have gradually been elucidated. These successes are partly due to the development of efficient expression and purification system of the membrane proteins including prestin. To obtain further understandings of prestin, the development of various types of such systems will be essential. However, recent study protocols on membrane proteins have often employed HEK293 cells and have become complexed with expression genes carrying several proteins and peptides for stabilization and purification of the expressed proteins. In the present study, a simple expression and purification system using Chinese hamster ovary (CHO) cells and Hi-tag was developed. MethodsFull length gerbil prestin was transfected into modified mammalian expression vectors with C-terminal 6 × His-tag. After drug selection with G418 for 4 weeks, single colonies were isolated by limiting dilution method. Cell lines highly expressing prestin were selected (named 3D5, 4D7 and 3C8). These cells were gently disrupted using a Dounce tissue grinder. Membrane fractions were extracted by ultracentrifugation and affinity chromatography was performed. The efficiency of the purification process was evaluated by quantitative Western blotting using a standard protein. ResultsAmong the cell lines constructed, Western blotting analysis showed bands at around 100 kDa and the highest intensity was confirmed from the 3C8 cell line, indicating that this cell line has the highest expression of prestin molecules. The membrane fraction was therefore extracted from this cell line and subjected to the following purification procedure. It was found that 78.7 μg of prestin was purified from 2.0 × 109 CHO cells. ConclusionIn the present study, 78.7 μg of prestin was purified from 2.0 × 109 CHO cells, which stably expressing 6 × His-tagged prestin, by extracting cell membrane fractions and standard affinity chromatography for His-tag.

Fast-diffusing receptor collisions with slow-diffusing peptide ligand assemble the ternary parathyroid hormone–GPCR–arrestin complex

The assembly of a peptide ligand, its receptor, and β-arrestin (βarr) into a ternary complex within the cell membrane is a crucial aspect of G protein-coupled receptor (GPCR) signaling. We explore this assembly by attaching fluorescent moieties to the parathyroid hormone (PTH) type 1 receptor (PTH1R), using PTH as a prototypical peptide hormone, along with βarr and clathrin, and recording dual-color single-molecule imaging at the plasma membrane of live cells. Here we show that PTH1R exhibits a near-Brownian diffusion, whereas unbound hormone displays limited mobility and slow lateral diffusion at the cell surface. The formation of the PTH–PTH1R–βarr complex occurs in three sequential steps: (1) receptor and ligand collisions, (2) phosphoinositide (PIP3)-dependent recruitment and conformational change of βarr molecules at the plasma membrane, and (3) collision of most βarr molecules with the ligand-bound receptor within clathrin clusters. Our results elucidate the non-random pathway by which PTH–PTH1R–βarr complex is formed and unveil the critical role of PIP3 in regulating GPCR signaling.

Cryo-EM structure of Nipah virus L-P polymerase complex

Nipah virus (NiV) is a non-segmented, negative-strand (NNS) RNA virus, belonging to Paramyxoviridae. The RNA polymerase complex, composed of large (L) protein and tetrameric phosphoprotein (P), is responsible for genome transcription and replication by catalyzing NTP polymerization, mRNA capping and cap methylation. Here, we determine the cryo-electron microscopy (cryo-EM) structure of fully bioactive NiV L-P polymerase complex at a resolution of 3.19 Å. The L-P complex displays a conserved architecture like other NNS RNA virus polymerases and L interacts with the oligomerization domain and the extreme C-terminus region of P tetramer. Moreover, we elucidate that NiV is naturally resistant to the allosteric L-targeting inhibitor GHP-88309 due to the conformational change in the drug binding site. We also find that the non-nucleotide drug suramin can inhibit the NiV L-P polymerase activity at both the enzymatic and cellular levels. Our findings have greatly enhanced the molecular understanding of NiV genome replication and transcription and provided the rationale for broad-spectrum polymerase-targeted drug design.

Site-specific incorporation of 19F-nulcei at protein C-terminus to probe allosteric conformational transitions of metalloproteins

Allosteric conformational change is an important paradigm in the regulation of protein function, which is typically triggered by the binding of small cofactors, metal ions or protein partners. Here, we found those conformational transitions can be effectively monitored by 19F NMR, facilitated by a site-specific 19F incorporation strategy at the protein C-terminus using asparaginyl endopeptidase (AEP). Three case studies show that C-terminal 19F-nuclei can reveal protein dynamics not only adjacent but also distal to C-terminus, including those occurring in a hemoprotein neuroglobin (Ngb), calmodulin (CaM), and a cobalt metalloregulator (CoaR) responding to both cobalt and tetrapyrrole. In Ngb, the heme orientation disorder is affected by missense mutations that perturb backbone rigidity or surface charges close to the heme axial ligands. In CaM, the C-terminal 19F-nuclei is an ideal probe for detecting the binding states of Ca2+, peptides and inhibitors. Furthermore, multiple 19F-moieties were incorporated into the two domains of CoaR, revealing the intrinsically disordered C-terminal metal binding tail might be an allosteric conformational switch to maintain cobalt homeostasis and balance corrinoid biosynthesis. This study demonstrates that the AEP-based 19F-modification strategy can be applied to various targets to study allosteric regulation, especially for those biological processes modulated by the protein C-terminus.

Phosphorylation-mediated conformational change regulates human SLFN11

Human Schlafen 11 (SLFN11) is sensitizing cells to DNA damaging agents by irreversibly blocking stalled replication forks, making it a potential predictive biomarker in chemotherapy. Furthermore, SLFN11 acts as a pattern recognition receptor for single-stranded DNA (ssDNA) and functions as an antiviral restriction factor, targeting translation in a codon-usage-dependent manner through its endoribonuclease activity. However, the regulation of the various SLFN11 functions and enzymatic activities remains enigmatic. Here, we present cryo-electron microscopy (cryo-EM) structures of SLFN11 bound to tRNA-Leu and tRNA-Met that give insights into tRNA binding and cleavage, as well as its regulation by phosphorylation at S219 and T230. SLFN11 phosphomimetic mutant S753D adopts a monomeric conformation, shows ATP binding, but loses its ability to bind ssDNA and shows reduced ribonuclease activity. Thus, the phosphorylation site S753 serves as a conformational switch, regulating SLFN11 dimerization, as well as ATP and ssDNA binding, while S219 and T230 regulate tRNA recognition and nuclease activity.

Role of Non-Binding T63 Alteration in IL-18 Binding

Engineered interleukin-18 (IL-18) has attracted interest as a cytokine-based treatment. However, knowledge-based mutagenesis of IL-18 has been reported for only a few regions of the protein structures, including binding sites I and II. When coupled with the binding region mutant (E6K), the non-binding residue of IL-18, Thr63 (T63), has been shown to increase the flexibility of the binding loop. Nevertheless, the function of Thr63 in conformational regulation is still unknown. Using homology modeling, molecular dynamics simulation, and structural analysis, we investigated the effects of Thr63 alteration coupling with E6K on conformational change pattern, binding loop flexibility, and the hydrogen bond network. The results indicate that the 63rd residue was significantly associated with hydrogen-bond relaxation at the core β-barrel binding sites I and II Glu85-Ile100 loop. This result provided conformational and flexible effects to binding sites I and III by switching their binding loops and stabilizing the 63rd residue cavity. These findings may pave the way for the conceptualization of a new design for IL-18 proteins by modifying non-binding residues for structure-based drug development.

AlphaFold2-Based Characterization of Apo and Holo Protein Structures and Conformational Ensembles Using Randomized Alanine Sequence Scanning Adaptation: Capturing Shared Signature Dynamics and Ligand-Induced Conformational Changes

Proteins often exist in multiple conformational states, influenced by the binding of ligands or substrates. The study of these states, particularly the apo (unbound) and holo (ligand-bound) forms, is crucial for understanding protein function, dynamics, and interactions. In the current study, we use AlphaFold2, which combines randomized alanine sequence masking with shallow multiple sequence alignment subsampling to expand the conformational diversity of the predicted structural ensembles and capture conformational changes between apo and holo protein forms. Using several well-established datasets of structurally diverse apo-holo protein pairs, the proposed approach enables robust predictions of apo and holo structures and conformational ensembles, while also displaying notably similar dynamics distributions. These observations are consistent with the view that the intrinsic dynamics of allosteric proteins are defined by the structural topology of the fold and favor conserved conformational motions driven by soft modes. Our findings provide evidence that AlphaFold2 combined with randomized alanine sequence masking can yield accurate and consistent results in predicting moderate conformational adjustments between apo and holo states, especially for proteins with localized changes upon ligand binding. For large hinge-like domain movements, the proposed approach can predict functional conformations characteristic of both apo and ligand-bound holo ensembles in the absence of ligand information. These results are relevant for using this AlphaFold adaptation for probing conformational selection mechanisms according to which proteins can adopt multiple conformations, including those that are competent for ligand binding. The results of this study indicate that robust modeling of functional protein states may require more accurate characterization of flexible regions in functional conformations and the detection of high-energy conformations. By incorporating a wider variety of protein structures in training datasets, including both apo and holo forms, the model can learn to recognize and predict the structural changes that occur upon ligand binding.

Quantifying conformational changes in the TCR:pMHC-I binding interface

BackgroundT cells form one of the key pillars of adaptive immunity. Using their surface bound T cell antigen receptors (TCRs), these cells screen millions of antigens presented by major histocompatibility complex (MHC) or MHC-like molecules. In other protein families, the dynamics of protein-protein interactions have important implications for protein function. Case studies of TCR:class I peptide-MHCs (pMHC-Is) structures have reported mixed results on whether the binding interfaces undergo conformational change during engagement and no robust statistical quantification has been done to generalise these results. Thus, it remains an open question of whether movement occurs in the binding interface that enables the recognition and activation of T cells.MethodsIn this work, we quantify the conformational changes in the TCR:pMHC-I binding interface by creating a dataset of 391 structures, comprising 22 TCRs, 19 MHC alleles, and 79 peptide structures in both unbound (apo) and bound (holo) conformations.ResultsIn support of some case studies, we demonstrate that all complementarity determining region (CDR) loops move to a certain extent but only CDR3α and CDR3β loops modify their shape when binding pMHC-Is. We also map the contacts between TCRs and pMHC-Is, generating a novel fingerprint of TCRs on MHC molecules and show that the CDR3α tends to bind the N-terminus of the peptide and the CDR3β tends to bind the C-terminus of the peptide. Finally, we show that the presented peptides can undergo conformational changes when engaged by TCRs, as has been reported in past literature, but novelly show these changes depend on how the peptides are anchored in the MHC binding groove.ConclusionsOur work has implications in understanding the behaviour of TCR:pMHC-I interactions and providing insights that can be used for modelling Tcell antigen specificity, an ongoing grand challenge in immunology.

Ligand response of guanidine-IV riboswitch at single-molecule level

Riboswitches represent a class of non-coding RNA that possess the unique ability to specifically bind ligands and, in response, regulate gene expression. A recent report unveiled a type of riboswitch, known as the guanidine-IV riboswitch, which responds to guanidine levels to regulate downstream genetic transcription. However, the precise molecular mechanism through which the riboswitch senses its target ligand and undergoes conformational changes remain elusive. This gap in understanding has impeded the potential applications of this riboswitch. To bridge this knowledge gap, our study investigated the conformational dynamics of the guanidine-IV riboswitch RNA upon ligand binding. We employed single-molecule fluorescence resonance energy transfer (smFRET) to dissect the behaviors of the aptamer, terminator, and full-length riboswitch. Our findings indicated that the aptamer portion exhibited higher sensitivity to guanidine compared to the terminator and full-length constructs. Additionally, we utilized Position-specific Labelling of RNA (PLOR) combined with smFRET to observe, at the single-nucleotide and single-molecule level, the structural transitions experienced by the guanidine-IV riboswitch during transcription. Notably, we discovered that the influence of guanidine on the riboswitch RNA’s conformations was significantly reduced after the transcription of 88 nucleotides. Furthermore, we proposed a folding model for the guanidine-IV riboswitch in the absence and presence of guanidine, thereby providing insights into its ligand-response mechanism.

Ligand response of guanidine-IV riboswitch at single-molecule level.

Riboswitches represent a class of non-coding RNA that possess the unique ability to specifically bind ligands and, in response, regulate gene expression. A recent report unveiled a type of riboswitch, known as the guanidine-IV riboswitch, which responds to guanidine levels to regulate downstream genetic transcription. However, the precise molecular mechanism through which the riboswitch senses its target ligand and undergoes conformational changes remain elusive. This gap in understanding has impeded the potential applications of this riboswitch. To bridge this knowledge gap, our study investigated the conformational dynamics of the guanidine-IV riboswitch RNA upon ligand binding. We employed single-molecule fluorescence resonance energy transfer (smFRET) to dissect the behaviors of the aptamer, terminator, and full-length riboswitch. Our findings indicated that the aptamer portion exhibited higher sensitivity to guanidine compared to the terminator and full-length constructs. Additionally, we utilized Position-specific Labelling of RNA (PLOR) combined with smFRET to observe, at the single-nucleotide and single-molecule level, the structural transitions experienced by the guanidine-IV riboswitch during transcription. Notably, we discovered that the influence of guanidine on the riboswitch RNA's conformations was significantly reduced after the transcription of 88 nucleotides. Furthermore, we proposed a folding model for the guanidine-IV riboswitch in the absence and presence of guanidine, thereby providing insights into its ligand-response mechanism.

Membrane tension-dependent conformational change of Isoleucine 106 of loop B diminishes water permeability in FaPIP2;1.

Aquaporins (AQPs) are membrane proteins specialized in facilitating water transport across membranes. Mechanical stress is one of the various stimuli that regulate AQPs. Briefly, there are several studies that report a decrease in permeability upon an increase in membrane tension. However, the molecular details of this mechanosensitive (MS) response are still a matter of debate. Our work attempts to close that gap in knowledge by providing evidence of a conformational change that occurs inside the pore of the strawberry aquaporin FaPIP2;1. Via osmotic shock experiments and molecular dynamics (MD) simulations, we found that a residue of loop B, I106, is key to the blocking of the permeation pathway and such a change is almost exclusively found under membrane tensile stress. In detail, osmotic shock experiments exhibited a nonlinear increment in water fluxes for increasing osmolarities, evidencing a decrease in the FaPIP2;1 permeability. MD simulations under membrane tension showed the same trend, with a significant increase in states with a low water permeability. The latter was correlated with a conformational change in I106 that generates a permeation barrier of around 18 kJ mol-1, effectively closing the pore. This work constitutes the first report of a PIP type aquaporin reacting to tensile stress in the membrane. Our findings could pave the way to test whether this conformational change is also responsible for mechanical gating in the other MS aquaporins, both those already reported and those still waiting to be found.

In silico Investigation of the Pro-apoptotic Potential of Syringic Acid Analog

Background: Conformational changes in BAX are associated with the activation of its pro-apoptotic potential. Previously, small molecule BAX antagonists have been shown to bring about apoptosis by inducing conformational changes in BAX by direct binding to the serine 184 site of BAX. Methods: In this article, we have proposed that syringic acid analog SA14 can incur apoptosis by directly binding to and inducing conformational changes in BAX. The pro-apoptotic potential of SA14 has been investigated using an in silico structure-based approach, i.e., docking and molecular dynamics computations are employed to study the binding of SA14 to the residues of the active site of BAX. Results: Based on docking results, four BAX-SA14 complexes, each representative of a cluster of conformations, have been selected for molecular dynamics simulations. The root mean square deviation has indicated the formation of stable conformations for two of the complexes. Other parameters, such as root mean square fluctuation, radius of gyration, and solvent accessible surface area, have been used to confirm the results, which have indicated favorable binding between BAX and SA14. Conclusion: Overall, the results have indicated that SA14 can bring about stable conformational changes in BAX and shows merit as a potential BAX-activating pro-apoptotic agent worthy of further experimental studies.

Abelmoschus manihot gum improves the water retention capacity of low-salt myofibrillar protein gels: Perspective on aggregation behaviour and conformational changes during heating

This study aimed to investigate the effect of Abelmoschus manihot gum (AMG) on the water retention capacity of low-salt myofibrillar protein (MP) gel by analysing its aggregation behaviour and conformational changes during heating (30–80 °C). The results revealed that AMG significantly increased the water holding capacity and facilitated the formation of a more uniform gel network structure in low-salt MP gel (P < 0.05). During the heat-induced gelation process, the solubility of low-salt MP significantly decreased, whereas its turbidity evidently increased as the level of added AMG increased (P < 0.05). Furthermore, the dynamic rheological behaviours indicated that low-salt MP-AMG gels underwent early denaturation and unfolded at 58 °C, finally forming an irreversible three-dimensional network at 80 °C. Moreover, adding AMG promoted α-helix-to-β-sheet transition in low-salt MP and decreased its fluorescence intensity during the heating process. Hydrophobic interactions and disulfide bonds were the two dominant forces governing the formation and maintenance of low-salt MP gel. The present study provides theoretical guidance for the production of novel low-salt healthy meat products.

Effect of dynamic high-pressure microfluidization on the structural, emulsifying properties, in vitro digestion and antioxidant activity of whey protein isolate.

The effects of dynamic high-pressure microfluidization (DHPM) on the structural, emulsifying properties, in vitro digestion and antioxidant activity of whey protein isolate (WPI) were investigated. The results demonstrated that WPI treated with 100MPa DHPM exhibited superior emulsification performance. This can be attributed to the conformational changes induced by 100MPa DHPM in WPI, leading to a transformation from disordered structures to ordered structures and an increased exposure of fluorophore such as tryptophan residues and hydrophobic groups, reduced aggregation state and particle size of WPI. These factors facilitated the migration of WPI towards the oil-water interface, resulting in the formation of a robust and compact adsorption layer which reduces interfacial tension and enhances emulsification stability. Furthermore, it was observed that while DHPM did not significantly affect the digestibility of WPI, it did enhance exposure to antioxidant amino acids in the digestive products thereby enhanced their antioxidant properties. In summary, structural modification induced by DHPM treatment enhanced both emulsification and antioxidant properties of WPI. These findings highlight the significant potential of DHPM treatment for enhancing the quality of meat products with an emulsion-type structure.

Design, Synthesis, Computational Studies and Evaluation of Novel 2, 4, 5-Trisubstituted Pyrimidine Derivatives for Anticancer Activity against MCF-7 and A549 Cell Lines

Synthesized novel trisubstituted pyrimidine derivatives (U 01-15) were tested for anti-cancer efficacy against two cancer cell line MCF-7 and A549. Several spectroscopic methods assisted to characterized and confirmed all synthesized compounds. Some of the synthesized pyrimidine derivatives showed strong effectiveness, with IC50 values comparable to the standard drug doxorubicin and gefitinib. Compounds U-02, U-04, U-07, U-10 and U-11 showed good anti-cancer activity for both cell lines having IC50 values in range from 12.06 µM to 22.45 µM, while compounds U-06 and U-03 showed moderate potency against both the cell lines, respectively. Using PyRx software (based on Auto Dock 4 and a genetic algorithm), all the synthesized compounds were put through a molecular docking with receptor tyrosine-protein kinase erbB-4 and docking score was compared to approved drugs, gefitinib and lapatinib. Interestingly almost all derivatives showed good docking score as compared to reference drugs. Molecular dynamics simulation of the U-11 protein-ligand complex over 100 ns revealed key insights into stability and conformational changes. RMSD analysis indicated stability between 10-20 ns with fluctuations up to 5.71 Å, while RMSF showed an average fluctuation of 1.74 Å, particularly in the loop regions. Molecular interactions, including hydrogen bonds, were observed with key residues such as ASP836, MET774, and LEU769.Future studies could focus on optimizing the most promising pyrimidine derivatives, such as U-02, U-04, U-07, U-10, and U-11, for enhanced potency and selectivity through advanced biological testing and structure-activity relationship studies may provide deeper insights into their therapeutic potential against cancer.

Receptor interactions of protoxin and activated Vip3Aa structural conformations in Spodoptera exigua.

The Vip3Aa insecticidal protein, produced by Bacillus thuringiensis, has been effectively used in commercial Bt-crops to manage lepidopteran pests. Upon ingestion by larvae, the protoxin is processed by midgut proteases into the activated protein and binds specifically to its receptors in the midgut, leading to insect mortality. Cryo-EM resolution of the trypsin-processed Vip3Aa protein unveiled structural remodelling of the N-terminal region during the transition from protoxin to activated protein. This conformational change has been demonstrated to be crucial for toxicity against Spodoptera exigua larvae, a major global lepidopteran pest. In this study, we investigated the relevance of the structural remodelling for the specific binding to midgut receptors. We conducted in vitro binding assays with radiolabelled proteins and brush border membrane vesicles (BBMV) from S. exigua, employing structural mutants that lock the protein in either its protoxin or its activated conformation. Our results indicate that both structural stages of the protein share binding sites in the midgut epithelium. Moreover, in vivo competition assays revealed that Vip3Aa is able to bind to functional receptors in S. exigua larvae both as protoxin and as activated protein. Altogether, our findings point to both structural conformations contributing to receptor binding. In vivo, either spontaneous structural shift upon proteolytic cleavage or receptor-mediated remodelling could be occurring. However, the timing and context in which the conformational change occurs could influence membrane insertion and toxicity. Our results show the complex interplay between proteolytic processing, protein structure and receptor interactions in Vip3Aa's toxicity. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

A pressure-jump EPR system to monitor millisecond conformational exchange rates of spin-labeled proteins.

Site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) using nitroxide spin labels is a well-established technology for mapping site-specific secondary and tertiary structure and for monitoring conformational changes in proteins of any degree of complexity, including membrane proteins, with high sensitivity. SDSL-EPR also provides information on protein dynamics in the timescale of ps-μs using continuous wave lineshape analysis and spin lattice relaxation time methods. However, the functionally important time domain of μs-ms, corresponding to large-scale protein motions, is inaccessible to those methods. To extend SDSL-EPR to the longer time domain, the perturbation method of pressure-jump relaxation is implemented. Here, we describe a complete high-pressure EPR system at Q-band for both static pressure and ms-timescale pressure-jump measurements on spin-labeled proteins. The instrument enables pressure jumps both up and down from any holding pressure, ranging from atmospheric pressure to the maximum pressure capacity of the system components (~3500 bar). To demonstrate the utility of the system, we characterize a local folding-unfolding equilibrium of T4 lysozyme. The results illustrate the ability of the system to measure thermodynamic and kinetic parameters of protein conformational exchange on the ms timescale.