Conformational Changes Research Articles

DNA-Based Molecular Clamp for Probing Protein Interactions and Structure under Force.

Cellular mechanotransduction, a process central to cell biology, embryogenesis, adult physiology, and multiple diseases, is thought to be mediated by force-driven changes in protein conformation that control protein function. However, methods to study proteins under defined mechanical loads on a biochemical scale are lacking. We report the development of a DNA-based device in which the transition between single- and double-stranded DNA applies tension to an attached protein. Using a fragment of the talin rod domain as a test case, negative-stain electron microscopy reveals programmable extension, while pull down assays show tension-induced binding to two ligands, ARPC5L and vinculin, known to bind to cryptic sites inside the talin structure. These results demonstrate the utility of the DNA clamp for biochemical studies and potential structural analysis.

Antibiofilm mechanism of 2R,3R-dihydromyricetin by targeting sortase A and its application against Staphylococcus aureus adhesion on eggshell

Biofilm formation of Staphylococcus aureus in food processing environments raises significant safety concerns, necessitating the development of new antibiofilm approaches for controlling S. aureus contamination. This study aimed to elucidate the antibiofilm mechanism of 2R,3R-dihydromyricetin (DMY), a natural flavonoid, against S. aureus and evaluate its efficacy in reducing bacterial adhesion to eggshell. The results revealed that DMY was a potent inhibitor of S. aureus sortase A (SrtA) with an IC50 of 73.43 μM, preventing bacterial adhesion to fibrinogen and subsequent biofilm formation. Fluorescence quenching assay and surface plasmon resonance analysis confirmed that DMY could directly bind to S. aureus SrtA. Notably, circular dichroism spectra demonstrated a conformational change in SrtA from α-helical to β-sheet structure upon DMY binding. Molecular dynamics simulation suggested that DMY bound to the catalytic pocket of S. aureus SrtA via hydrophobic interactions and hydrogen bonds. Furthermore, fluorescence microscopic observations further revealed that DMY attenuated the biofilm-related phenotype of SrtA by decreasing the anchoring of S. aureus protein A (SpA) onto cell wall. Importantly, pretreatment with 125 μg/mL DMY significantly reduced 1.14–1.75 log CFU/cm2 of S. aureus adhered on eggshells. Overall, these findings highlight how specific targeting of SrtA by DMY inhibits the attachment stages of biofilm development in S. aureus, making it a promising candidate for a novel disinfectant against this pathogen in the food industry.

Influence of Wobbling Tryptophan and Mutations on PET Degradation Explored by QM/MM Free Energy Calculations.

Plastic-degrading enzymes, particularly poly(ethylene terephthalate) (PET) hydrolases, have garnered significant attention in recent years as potential eco-friendly solutions for recycling plastic waste. However, understanding of their PET-degrading activity and influencing factors remains incomplete, impeding the development of uniform approaches for enhancing PET hydrolases for industrial applications. A key aspect of PET hydrolase engineering is optimizing the PET-hydrolysis reaction by lowering the associated free energy barrier. However, inconsistent findings have complicated these efforts. Therefore, our goal is to elucidate various aspects of enzymatic PET degradation by means of quantum mechanics/molecular mechanics (QM/MM) reaction simulations and analysis, focusing on the initial reaction step, acylation, in two thermophilic PET hydrolases, LCC and PES-H1, along with their highly active variants, LCCIG and PES-H1FY. Our findings highlight the impact of semiempirical QM methods on proton transfer energies, affecting the distinction between a two-step reaction involving a metastable tetrahedral intermediate and a one-step reaction. Moreover, we uncovered a concerted conformational change involving the orientation of the PET benzene ring, altering its interaction with the side-chain of the "wobbling" tryptophan from T-stacking to parallel π-π interactions, a phenomenon overlooked in prior research. Our study thus enhances the understanding of the acylation mechanism of PET hydrolases, in particular by characterizing it for the first time for the promising PES-H1FY using QM/MM simulations. It also provides insights into selecting a suitable QM method and a reaction coordinate, valuable for future studies on PET degradation processes.

Uncovering conserved networks and global conformational changes in G protein-coupled receptor kinases

G protein-coupled receptor kinases (GRKs) are essential regulators of signaling pathways mediated by G protein-coupled receptors. Recent research suggests that GRK-mediated phosphorylation patterns dictate functional selectivity, leading to biased cellular responses. However, a comprehensive understanding of the structural mechanisms at the single-residue level remains elusive. This study aims to define the general conformational dynamics of GRKs with a particular focus on quantifying the transitions between the closed and open states. Specifically, we examined these transitions, classified based on the ionic lock between the regulatory G protein signaling homology domain and kinase domain. To facilitate a precise structural comparison, we assigned common labels to topologically identical positions across the 47 GRK structures retrieved from the Protein Data Bank. Our analysis identified both general and subfamily-specific dynamic movements within the networks and measured the conformational change scores between the two states. Elucidating these structural dynamics could provide significant insights into the regulatory mechanisms of GRK.

Regulatory effects of cyclodextrins on light-responsive phase transition behaviors of poly(N-isopropylacrylamide-co-N-(4-phenylazophenyl)methylacrylamide)

The physicochemical properties of light-responsive polymers have attracted widespread attention due to their ability to be precisely and conveniently modulated by light without additives or contact. Here, we report a thermo/light dual-responsive polymer, PNMA, synthesized through the free radical copolymerization of azobenzene derivative (mAzo) and N-isopropylacrylamide (NIPAM) monomers. By switching between 365 nm UV light and 450 nm visible light, the E/Z photoisomerization of the light-responsive mAzo can be reversibly controlled, and thereby adjusting the conformational changes of the PNMA polymer chain. Significantly, the stretching and contraction conformations of PNMA polymer chains can be manipulated by incorporating different types of cyclodextrins. A comprehensive comparative study was conducted to determine the light-controlled binding and dissociation processes between the azobenzene units and CDs (α-CD, β-CD and γ-CD), the influences of CDs on the photoisomerization kinetics and complexation constants of azobenzene with CDs, as well as the effect of CDs on the light-responsive phase transition behaviors of PNMA. Our results highlight the superior advantage of α-CD in constructing light-responsive systems with significant changes in hydrophilicity. Notably, by tuning the concentration of α-CD and the proportion of mAzo in PNMA, the range of light-induced thermosensitive response is significantly expanded. This study is the first to present a comparative study on the effects of three CDs on the thermo/light dual-responsive properties of polymer systems based on PNIPAM and azobenzene, providing new ideas for CD selection in the design and construction of advanced light-responsive materials based on light-responsive guest-host dissociation or hydrophilic changes of azobenzene/CD complexes.

Biased Signaling Agonists Promote Distinct Phosphorylation and Conformational States of the Dopamine D3 Receptor

Biased agonists of G-protein-coupled receptors (GPCRs) have emerged as promising selective modulators of signaling pathways by offering therapeutic advantages over unbiased agonists to minimize side effects. The dopamine D3 receptor (D3R), a pivotal GPCR in the central nervous system, has gained significant attention as a therapeutic target for neurological diseases, including Parkinson’s disease (PD), addiction, psychosis, depression, and anxiety. We have recently designed and tested SK609, a G-protein biased D3R selective agonist, and demonstrated its efficacy in reducing motor impairment and improving cognitive effects in a rodent model of PD. The molecular mechanism by which SK609 recruits G-protein but not β-arrestin pathways is poorly understood. Utilizing all-atom molecular dynamics simulations, we investigated the distinct conformational dynamics imparted by SK609 and the reference unbiased agonist Pramipexole (PRX). Results from these studies show that the flexibility of transmembrane 3 is key to unbiased signaling, with a ~30° and ~17° shift in tilt angle in the D3R-Gi and D3R-βarrestin2 complexes, respectively. Additionally, untargeted phosphoproteomics analysis reveals unique phosphorylation sites by SK609 and PRX in D3R. These results suggest that SK609 induces conformational changes and unique phosphorylation patterns that promote interactions with G-proteins and are not conducive for β-arrestin2 recruitment and signaling.

A molecular mechanism for how pressure induces interdigitation of phospholipid bilayer membranes

The phase transition from the ripple gel phase to the interdigitated gel phase of bilayers of phosphatidylcholines (PCs) with two saturated long-chain fatty acids under high pressure was investigated by pressure-scanning microscopy, fluorometry, and dynamic light scattering (DLS) measurements. Microscopic observation for giant vesicles (GVs) of distearoyl-PC (DSPC) under high pressure showed that spherical GVs transforms significantly into warped and distorted spherical ones instantaneously at the pressure-induced interdigitation. The fluorescence intensities of amphiphilic probe Prodan and hydrophobic probe Laurdan in the dipalmitoyl-PC (DPPC) bilayer steeply decreased and increased, respectively, at the interdigitation, suggesting that the conformational change of the polar head group of DPPC molecule in the bilayer transiently occurred at the interdigitation. Further, it was found from the high-pressure DLS measurements that the size of the vesicle particles of the DPPC and DSPC transiently increases near the interdigitation pressure, whereas the chemically induced interdigitation by adding ethanol to the DSPC bilayer membrane under atmospheric pressure produce no such change in the particle size. Taking account of the critical packing parameter of the PC molecule, the above experimental results would lead us to the conclusion that the pressure-induced interdigitation is attributable to the increase in repulsive interaction between the polar head groups of the PC molecules resulting from the orientational change of the head group from a parallel alignment to a perpendicular one with respect to the bilayer surface by applying pressure, namely the transient state: it occurs when the repulsive interaction exceeds a threshold value for the balance between the repulsive interaction and the attractive interaction among the hydrophobic acyl chains.

Investigating the interaction mechanisms between arachin and resveratrol: Utilizing multi-spectroscopy and computational chemistry

Arachin (ARA) and resveratrol (RES) are the primary protein and bioactive compound in peanuts and their processed products. However, the mechanism of interaction between these two substances remained unclear. To investigate protein structural changes, conformational variations, and molecular mechanisms in the interaction between them, multispectral analysis and computational chemistry methods were employed. Experimental results confirmed that RES quenched ARA's intrinsic fluorescence through static quenching, indicating their interaction. Thermodynamic analysis revealed the interaction between them was endothermic, spontaneous, and primarily hydrophobic. Molecular dynamics (MD) simulations highlighted strong affinity between RES and ARA, with key amino acids (His425, Val426, Phe405, and Phe464) facilitating their interaction. RES binding increased stability without significant protein conformational changes. The independent gradient model based on Hirshfeld partition (IGMH) validated their interaction, emphasizing van der Waals (VDW) interactions and hydrogen bonds (H-bonds) as crucial for stable binding. This research lays a theoretical foundation for potential applications of ARA-RES complex products in the food industry.

L-lysine enhances pork color through antioxidant activity and myoglobin conformational changes

This study aimed to investigate the effect of L-lysine (Lys) on the color of pork and reveal the possible mechanism. The results showed that the L* and a* values increased from 53.69 to 56.32, 56.39, and 56.47, and from 12.93 to 13.21, 13.24, and 13.52 with the addition of 0.1 %, 0.15 % and 0.2 % Lys, respectively. Meanwhile, the oxymyoglobin (Fe2+) levels increased from 21.14 % to 22.63 %, 23.83 %, and 23.53 %, while the metmyoglobin (Fe3+) levels decreased from 44.69 % to 40.28 %, 41.21 %, and 41.63 % with the addition of 0.1 %, 0.15 % and 0.2 % Lys, respectively. Additionally, the addition of Lys increased total sulfhydryl and active sulfhydryl contents, and decreased the levels of reactive oxygen species (ROS) (P<0.05). The particle size and the absolute value of the ζ-potential increased with the addition of Lys, reaching maximum values of 534.39 nm and −13.73 mV, respectively. The molecular dynamics results suggested that Lys can bind to myoglobin (Mb) through hydrophobic interactions and hydrogen bonds, forming a stable Lys-Mb complex, acting as a protective shield to prevent the entry of ROS and other oxidizing agents. Finally, the addition of 0.15 % Lys resulted in the highest surface hydrophobicity, which was 5.79 μg. The multispectral results indicated that Lys primarily induces changes in the secondary and tertiary structures of Mb through interactions with tyrosine residues. These changes stabilized the free-moving rings within the amino acid residues of Mb, thereby improving the structural stability of Mb and ultimately enhancing the color stability of pork. In summary, Lys improved meat color stability through a dual mechanism of antioxidation and interaction with Mb to alter its conformational stability.

A Method for Treating Significant Conformational Changes in Alchemical Free Energy Simulations of Protein-Ligand Binding.

Relative binding free energy (RBFE) simulation is a rigorous approach to the calculation of quantitatively accurate binding free energy values for protein-ligand binding in which a reference binder is gradually converted to a target binder through alchemical transformation during the simulation. The success of such simulations relies on being able to accurately sample the correct conformational phase space for each alchemical state; however, this becomes a challenge when a significant conformation change occurs between the reference and target binder-receptor complexes. Increasing the simulation time and using enhanced sampling methods can be helpful, but effects can be limited, especially when the free energy barrier between conformations is high or when the correct target complex conformation is difficult to find and maintain. Current RBFE protocols seed the reference complex structure into every alchemical window of the simulation. In our study, we describe an improved protocol in which the reference structure is seeded into the first half of the alchemical windows, and the target structure is seeded into the second half of the alchemical windows. By applying information about the relevant correct end point conformations to different simulation windows from the beginning, the need for large barrier crossings or simulation prediction of the correct structures during an alchemical simulation is in many cases obviated. In the diverse cases we examine below, the simulations yielded free energy predictions that are satisfactory as compared to experiment and superior to running the simulations utilizing the conventional protocol. The method is straightforward to implement for publicly available FEP workflows.

Near-Infrared Ratiometric Hemicyanine Fluorescent Probes for Monitoring Mitochondrial pH Dynamics in Live Cells during Oxidative Stress and Hypoxia.

Novel near-infrared ratiometric molecules (probes A and B) produced by linking formyl-functionalized xanthene and methoxybenzene moieties, respectively, onto a xanthene-hemicyanine framework are detailed. Probe A exhibited a primary absorption peak at 780 nm and a shoulder peak at 730 nm and exhibited fluorescence at 740 nm↓ (signifies a downward shift in intensity upon acidification) in a pH 9.3 buffer and 780 nm↑ at pH 2.8 under excitation at 700 nm. Probe B featured absorptions at 618 and 668 nm at pH 3.2 and at 717 nm at pH 8.6, and fluorescence at 693 nm↑ at pH 3.2 and at 739 nm↓ at pH 8.6, in mostly the red to near-IR region. The ratiometric changes in the intensity of the fluorescent absorptions were reversed between A and B upon acidification as indicated by the arrows. Theoretical calculations confirmed that there were slight changes in conformation between probes and the protonated molecules, suggesting that the changes in emission spectra were due mostly to conjugation effects. Calculations at the APFD/6-311+g(d,p) level with a solvent described by the polarizable continuum model resulted in pK a values for A at 6.33 and B at 6.41, in good agreement with the experimentally determined value of 6.97 and an average of 6.40, respectively. The versatilities of the probes were demonstrated in various experimental contexts, including the effective detection of mitochondrial pH fluctuations. Live cell experiments involving exposure to different pH buffers in the presence of H+ ionophores, monitoring mitophagy processes during cell starvation, studying hypoxia induced by CoCl2 treatment, and investigating responses to various oxidative stresses are detailed. Our findings highlight the potential of attaching xanthene and methoxybenzaldehyde groups onto xanthene-hemicyanine structures as versatile tools for monitoring pH changes in a variety of cellular environments and processes.

Probing the interaction of hesperidin showing antiproliferative activity in colorectal cancer cells and human hemoglobin

Hesperidin, a flavanone glycoside abundant in citrus is known to possess anti-carcinogenic properties. However, its main interaction with cancer cells and blood proteins is not well-studied yet. Here we have explored the interactions of hesperidin with human colorectal cancer cells, HCT116, and human hemoglobin (HHb) with several experimental and theoretical studies. Cellular assays showed that hesperidin interacted with colorectal cancer cells and induced membrane damage, colony formation inhibition, oxidative stress, mitochondrial dysfunction, Bax/Bcl-2, caspase-9, and caspase-3 upregulation, and cytochrome c release determined by cellular, qPCR and ELISA assays. The interaction of the hesperidin with HHb indicated the formation of a static complex mainly with the assistance of hydrogen bonds which lead to partial folding of protein determined by spectroscopy, molecular docking, and molecular dynamic studies. In conclusion, these findings show that hesperidin with potential binding affinity with a plasma protein model can also show promising anticancer activities against colorectal cancer cells.

A Review of Electromagnetic Fields in Cellular Interactions and Cacao Bean Fermentation.

The influence of magnetic fields on biological systems, including fermentation processes and cocoa bean fermentation, is an area of study that is under development. Mechanisms, such as magnetosensitivity, protein conformational changes, changes to cellular biophysical properties, ROS production, regulation of gene expression, and epigenetic modifications, have been identified to explain how magnetic fields affect microorganisms and cellular processes. These mechanisms can alter enzyme activity, protein stability, cell signaling, intercellular communication, and oxidative stress. In cacao fermentation, electromagnetic fields offer a potential means to enhance the sensory attributes of chocolate by modulating microbial metabolism and optimizing flavor and aroma development. This area of study offers possibilities for innovation and the creation of premium food products. In this review, these aspects will be explored systematically and illustratively.

Exploring phosphorene-protein interactions: An integrated computational and spectroscopic investigation

Among 2D materials, exfoliated black phosphorus (or phosphorene) shows great promise for applications in biological domains. However, despite its performances, little is known about the intricate and dynamic interactions that this material can form with proteins. This increases the risk of off-target effects and adds complexity in designing phosphorene-based devices with tailored properties. In this study, we present a straightforward and easily implementable pipeline that integrates spectroscopies with Molecular Dynamics simulations to explore the dynamic interplay between phosphorene and a protein system. Using lysozyme as a deeply investigated reference protein, we employed two theoretical protein models with unique secondary structure folds to increase the descriptive power of the approach and disentangle the complexity and variability of experimental data into a few primary drivers of protein-phosphorene interactions. Our results show that the 2D material does not significantly alter the protein structure, but the observed conformational changes are influenced by the secondary fold. Indeed, while the beta structure interacts mainly through unfolded regions, the alpha fold favours phosphorene binding through structured clusters of residues, leading to more significant structural and dynamic perturbations. By utilizing this pipeline, we have gained valuable insights into the molecular recognition mechanism of phosphorene, enhancing the development of improved phosphorene-based devices. In addition, our methodology offers potential for further applications in biomedicine to characterise interfaces between other 2D (nano)materials and biological entities.

Inhibition of Key Virulence Enzymes of Botrytis cinerea by Flavonoids from the Antarctic Plant Colobanthus quitensis.

This study investigates the antifungal efficacy of flavonoids derived from Colobanthus quitensis against key virulence-related enzymes implicated in the pathogenic mechanisms of Botrytis cinerea. The flavonoids swertiajaponin, schaftoside, vitexin, and saponarin significantly inhibited pectinase, cellulase, and laccase activity. Specifically, swertiajaponin showed mixed inhibition of pectinase and cellulase, characterized by high affinity (low inhibition constant -Ki-) for enzyme-substrate complexes. Schaftoside showed mixed inhibition of pectinase and competitive inhibition of laccase, effectively reducing enzymatic activity by competing directly with the substrate. In contrast, vitexin showed competitive inhibition of pectinase and non-competitive inhibition of laccase, suggesting it induces conformational changes within the enzyme. Finally, saponarin uniquely showed uncompetitive inhibition of laccase, stabilizing the enzyme-substrate complex and thereby markedly reducing catalytic turnover. Supported by kinetic parameters (maximum velocity -Vmax-, Michaelis constant -Km-, and Ki), these findings highlight the potential of flavonoids from C. quitensis as natural fungicides, offering a sustainable and eco-friendly alternative to synthetic fungicides for managing agricultural diseases.

Phosphorylation at the D56 residue of MtrA in Mycobacterium tuberculosis enhances its DNA binding affinity by modulating inter-domain interaction

The response regulator, MtrA, plays a major role in adaptation to the host environment, cell division, replication, and dormancy activation of Mycobacterium tuberculosis (Mtb). The phosphorylation of the response regulator MtrA alters the downstream activity, typically involving changes in DNA binding activity. However, there is a substantial knowledge gap in understanding the phosphorylation-mediated structural changes in MtrA. Additionally, the active conformation of the protein has yet to be determined. Therefore, in this study, we have investigated the phosphorylation-induced conformational changes of MtrA using all-atom molecular dynamics simulations under various phosphorylation conditions. The results from this study demonstrate that the phosphorylation at D56 (pD56-MtrA) increases the compactness of the MtrA protein by stabilizing the inter-domain interaction between the regulatory domain and DNA binding domain. Notably, the higher occupancy H-bond (over 95 %) between Arg200-Asn100 in case of the pD56-MtrA condition, which is otherwise absent in the non-phosphorylated (uMtrA) condition, suggests the importance of this interaction in the active conformation of the protein. The dynamic cross-correlation analysis reveals that phosphorylation (especially pD56-MtrA) reduces the anti-correlated motions and increases correlated motions between different domains. Moreover, the higher DNA binding affinity of pD56-MtrA compared to uMtrA supported by molecular docking and MD simulation followed by MMPBSA analysis suggests that pD56-MtrA is the possible active conformation of the MtrA protein. Overall, this investigation elucidates the key structural changes in MtrA under different phosphorylated conditions, which might help in designing novel therapeutics against tuberculosis.

Antagonistic nanobodies implicate mechanism of GSDMD pore formation and potential therapeutic application

Inflammasome activation results in the cleavage of gasdermin D (GSDMD) by pro-inflammatory caspases. The N-terminal domains (GSDMDNT) oligomerize and assemble pores penetrating the target membrane. As methods to study pore formation in living cells are insufficient, the order of conformational changes, oligomerization, and membrane insertion remained unclear. We have raised nanobodies (VHHs) against human GSDMD and find that cytosolic expression of VHHGSDMD-1 and VHHGSDMD-2 prevents oligomerization of GSDMDNT and pyroptosis. The nanobody-stabilized GSDMDNT monomers partition into the plasma membrane, suggesting that membrane insertion precedes oligomerization. Inhibition of GSDMD pore formation switches cell death from pyroptosis to apoptosis, likely driven by the enhanced caspase-1 activity required to activate caspase-3. Recombinant antagonistic nanobodies added to the extracellular space prevent pyroptosis and exhibit unexpected therapeutic potential. They may thus be suitable to treat the ever-growing list of diseases caused by activation of (non-) canonical inflammasomes.

Photo-thermal effects initiate multi-level energy conversion in “solid-solid” phase-changing fibers

The current textiles primarily employ passive heat barriers to minimize heat loss and achieve effective thermal insulation for human beings. Accordingly, intelligent fibers with energy storage and temperature control capabilities have garnered significant attention due to their potential to revolutionize textile technology. The study integrates the photo-thermal effect and phase change energy storage materials onto a fiber, thereby fabricating a fully intelligent energy storage fiber. This innovation enables the multi-level conversion of sunlight: “Optical energy - Thermal energy - Phase transition energy - Thermal energy”. The intelligent fiber efficiently converts solar energy into heat energy through the photo-thermal coupling of CuNPs, subsequently inducing a spatial conformational change in the solid-solid phase change material within the fiber for effective heat storage. The hybrid fiber possesses enhanced mechanical properties but also exhibits a significantly high phase transition enthalpy value of 49.75 J g−1 and a phase transition temperature suitable for human body temperature (20.19–30.21 °C), especially the fiber is more durable. The photo-thermal conversion test vividly demonstrates the systematic transformation of four distinct forms of energy within the composite fiber. This approach holds significant potential for advancing the field of smart fiber technology.

Cellular Prion Protein Conformational Shift after Liquid-Liquid Phase Separation Regulated by a Polymeric Antagonist and Mutations.

Liquid-liquid phase separation (LLPS) of intrinsically disordered proteins has been associated with neurodegenerative diseases, although direct mechanisms are poorly defined. Here, we report on a maturation process for the cellular prion protein (PrPC) that involves a conformational change after LLPS and is regulated by mutations and poly(4-styrenesulfonic acid-co-maleic acid) (PSCMA), a molecule that has been reported to rescue Alzheimer's disease-related cognitive deficits by antagonizing the interaction between PrPC and amyloid-β oligomers (Aβo). We show that PSCMA can induce reentrant LLPS of PrPC and lower the saturation concentration (Csat) of PrPC by 100-fold. Regardless of the induction method, PrPC molecules subsequently undergo a maturation process to restrict molecular motion in a more solid-like state. The PSCMA-induced LLPS of PrPC stabilizes the intermediate LLPS conformational state detected by NMR, though the final matured β-sheet-rich state of PrPC is indistinguishable between induction conditions. The disease-associated E200 K mutation of PrPC also accelerates maturation. This post-LLPS shift in protein conformation and dynamics is a possible mechanism of LLPS-induced neurodegeneration.

Molecular Dynamic Simulations Reveal that Water-Soluble QTY-Variants of Glutamate Transporters EAA1, EAA2 and EAA3 Retain the Conformational Characteristics of Native Transporters

ObjectiveGlutamate transporters play a crucial role in neurotransmitter homeostasis, but studying their structure and function is challenging due to their membrane-bound nature. This study aims to investigate whether water-soluble QTY-variants of glutamate transporters EAA1, EAA2 and EAA3 retain the conformational characteristics and dynamics of native membrane-bound transporters.MethodsMolecular dynamics simulations and comparative genomics were used to analyze the structural dynamics of both native transporters and their QTY-variants. Native transporters were simulated in lipid bilayers, while QTY-variants were simulated in aqueous solution. Lipid distortions, relative solvent accessibilities, and conformational changes were examined. Evolutionary conservation profiles were correlated with structural dynamics. Statistical analyses included multivariate analysis to account for confounding variables.ResultsQTY-variants exhibited similar residue-wise conformational dynamics to their native counterparts, with correlation coefficients of 0.73 and 0.56 for EAA1 and EAA3, respectively (p < 0.001). Hydrophobic interactions of native helices correlated with water interactions of QTY- helices (rs = 0.4753, p < 0.001 for EAA1). QTY-variants underwent conformational changes resembling the outward-to-inward transition of native transporters.ConclusionsWater-soluble QTY-variants retain key structural properties of native glutamate transporters and mimic aspects of native lipid interactions, including conformational flexibility. This research provides valuable insights into the conformational changes and molecular mechanisms of glutamate transport, potentially offering a new approach for studying membrane protein dynamics and drug interactions.