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Abstract Metabotropic glutamate receptor 2 (mGluR2), a subclass C member of the G protein-coupled receptor (GPCR) superfamily, is essential for regulating neurotransmitter signaling and facilitating synaptic adaptability in the central nervous system. This receptor, like other GPCRs, is highly sensitive to its surrounding lipid environment, where specific lipid compositions can influence its stability, conformational dynamics, and function. In particular, cholesteryl hemisuccinate (CHS) plays a critical role in stabilizing mGluR2 and modulating its structural states within cellular membranes and micellar environments. However, the molecular basis for this lipid-mediated modulation remains largely unexplored. To investigate the effects of CHS and lipid composition on mGluR2, we employed all-atom molecular dynamics simulations of mGluR2 embedded in both detergent micelles (BLMNG and CHS) and a POPC lipid bilayer containing 0%, 10%, and 25% CHS. These simulations were conducted for both active and inactive states of the receptor. Our findings reveal that CHS concentration modulates mGluR2’s structural stability and conformational behavior, with a marked impact observed within transmembrane helices TM1, TM2, and TM3, which constitute the core of the receptor’s transmembrane domain. In micelle environments, mGluR2 displayed unique conformational dynamics influenced by CHS, underscoring the receptor’s sensitivity to its lipid surroundings. Notably, a CHS concentration of 10% elicited more pronounced conformational changes than either cholesterol-depleted (0%) or cholesterol-enriched (25%) systems, indicating an optimal CHS range for maintaining structural stability. Our study provides atomistic insights into how lipid composition and CHS concentration impact mGluR2’s conformational landscape in distinct micelle and bilayer environments. These findings advance our understanding of lipid-mediated modulation in GPCR function, highlighting potential avenues for receptor-targeted drug design, particularly in cases where lipid interactions play a significant role in therapeutic efficacy. 

Abstract Liposomal carriers provide a flexible and effective strategy for delivering therapeutics across a broad spectrum of diseases. Cholesterol is frequently included in these systems to improve membrane rigidity and limit permeability. Despite its widespread use, the optimal cholesterol-to-lipid proportion for achieving stable and efficient liposome performance remains to be fully determined. In this work, we apply all-atom molecular dynamics simulations to explore how different cholesterol concentrations influence the structural and dynamic characteristics of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) bilayers, considering both planar and curved membrane geometries. Bilayers with cholesterol molar ratios of 0%, 10%, 20%, 30%, 40%, and 50% were simulated, and key biophysical parameters including area per lipid (APL), membrane thickness, leaflet interdigitation, and deuterium order parameters (SCD) were analyzed. In planar bilayers, increasing cholesterol concentration led to a progressive decrease in APL from approximately 60^°A²to 40^°A², accompanied by increased membrane thickness and lipid ordering, consistent with cholesterol’s classical condensing effect. In contrast, curved bilayers exhibited a cholesterol-induced expansion effect, particularly in the inner leaflet, where APL increased from approximately 60^°A²to 90^°A²with rising cholesterol levels. SCD profiles showed that cholesterol enhanced tail ordering up to 40% concentration, beyond which the effect plateaued or slightly declined, suggesting structural saturation or packing frustration. Membrane thickness displayed a monotonic increase in planar bilayers but followed a nonlinear trend in curved systems due to curvature-induced stress. These findings highlight that cholesterol’s influence on membrane properties is highly dependent on bilayer geometry and asymmetry. While planar bilayers exhibit predictable responses, curved systems reveal nonclassical behaviors that challenge traditional models of cholesterol-lipid interactions. This work provides molecular-level insights and establishes a computational framework for the rational design of liposomal systems, emphasizing the need to account for curvature and asymmetry in membrane engineering. 

Abstract Sav1866, a bacterial ATP-binding cassette (ABC) exporter, plays a crucial role in cellular processes by facilitating the efflux of a diverse range of substrates, including drugs, chemotherapeutic agents, peptides, and lipids. This efflux activity significantly impacts the effectiveness of various therapies against bacterial infections. In our recent investigation, we focused on understanding the conformational dynamics of Sav1866 within different lipid environments. Specifically, we explored its behavior in environments composed of DMPC and POPE lipids, which exhibit crucial distinctions not only in their headgroup polarity but also in the length and saturation of their hydrophobic tails. Our extensive set of equilibrium microsecond-level all-atom molecular dynamics (MD) simulations revealed significant distinctions in transporter behavior influenced by these lipid compositions. We observed a rapid transition to an occluded-inward-facing (IF-occ) conformation in POPE environments, contrasting with a channel-like behavior in DMPC environments, deviating from the expected alternating access mechanism (AAM). These findings underscore the significant impact of lipid compositions on ABC transporter function, offering new perspectives on membrane transport mechanisms. 

Abstract Metabotropic glutamate receptors (mGluRs) are class C G protein-coupled receptors that function as obligate dimers in regulating neurotransmission and synaptic plasticity in the central nervous system. The mGluR1 subtype has been shown to be modulated by the membrane lipid environment, particularly cholesterol, though the molecular mechanisms remain elusive. In this study, we employed all-atom molecular dynamics simulations to investigate the effects of cholesterol on the conformational dynamics of the mGluR1 seven-transmembrane (7TM) domain in an inactive state model. Simulations were performed with three different cholesterol concentrations (0%, 10%, and 25%) in a palmitoyl-oleoyl phosphatidylcholine (POPC) lipid bilayer system. Our results demonstrate that cholesterol induces conformational changes in the mGluR1 dimer more significantly than in the individual protomers. Notably, cholesterol modulates the dynamics and conformations of the TM1 and TM2 helices at the dimer interface. Interestingly, an intermediate cholesterol concentration of 10% elicits more pronounced conformational changes compared to both cholesterol-depleted (0%) and cholesterol-enriched (25%) systems. Specific electrostatic interaction unique to the 10% cholesterol system further corroborate these conformational differences. Given the high sequence conservation of the 7TM domains across mGluR subtypes, the cholesterol-dependent effects observed in mGluR1 are likely applicable to other members of this receptor family. Our findings provide atomistic insights into how cholesterol modulates the conformational landscape of mGluRs, which could impact their function and signaling mechanisms. 

Abstract The protein–ligand binding affinity quantifies the binding strength between a protein and its ligand. Computer modeling and simulations can be used to estimate the binding affinity or binding free energy using data- or physics-driven methods or a combination thereof. Here we discuss a purely physics-based sampling approach based on biased molecular dynamics simulations. Our proposed method generalizes and simplifies previously suggested stratification strategies that use umbrella sampling or other enhanced sampling simulations with additional collective-variable-based restraints. The approach presented here uses a flexible scheme that can be easily tailored for any system of interest. We estimate the binding affinity of human fibroblast growth factor 1 to heparin hexasaccharide based on the available crystal structure of the complex as the initial model and four different variations of the proposed method to compare against the experimentally determined binding affinity obtained from isothermal titration calorimetry experiments. 

Abstract Human acidic fibroblast growth factor (hFGF1) is an all beta-sheet protein that is involved in the regulation of key cellular processes including cell proliferation and wound healing. hFGF1 is known to aggregate when subjected to thermal unfolding. In this study, we investigate the equilibrium unfolding of hFGF1 using a wide array of biophysical and biochemical techniques. Systematic analyses of the thermal and chemical denaturation data on hFGF1 variants (Q54P, K126N, R136E, K126N/R136E, Q54P/K126N, Q54P/R136E, and Q54P/K126N/R136E) indicate that nullification of charges in the heparin-binding pocket can significantly increase the stability of wtFGF1. Triple variant (Q54P/K126N/R136E) was found to be the most stable of all the hFGF1 variants studied. With the exception of triple variant, thermal unfolding of wtFGF1 and the other variants is irreversible. Thermally unfolded triple variant refolds completely to its biologically native conformation. Microsecond-level molecular dynamic simulations reveal that a network of hydrogen bonds and salt bridges linked to Q54P, K126N, and R136E mutations, are responsible for the high stability and reversibility of thermal unfolding of the triple variant. In our opinion, the findings of the study provide valuable clues for the rational design of a stable hFGF1 variant that exhibits potent wound healing properties. 

Removing glutamate from the synaptic cleft is vital for proper function of the brain. Excitatory amino acid transporters mediate this process by uptaking the neurotransmitter from the synaptic cleft back to the cell after its release. The archaeal homolog, Glt_Ph, an aspartate transporter fromPyrococcus horikoshii, presents the best structurally characterized model for this family of transporters. In order to transport, Glt_Phundergoes elevator-like conformational changes between inward-facing (IF) and outward-facing (OF) states. Here, we characterize, at an atomic level, the OF⇌IF transition of Glt_Phin differentapo/bound states using a combination of ensemble-based enhanced sampling techniques, employing more than two thousand of coupled simulation replicas of membrane-embedded Glt_Ph. The resulting free-energy profiles portray the transition ofapo/bound states as a complex four-stage process, while sodium binding alone locks the structure in one of its states. Along the transition, the transport domain (TD) disengages from the scaffold domain (SD), allowing it to move as a piston sliding vertically with respect to the membrane during the elevator-like motion of TD. Lipid interactions with residues comprising the SD–TD interface directly influence the large-scale conformational changes and, consequently, the energetics of transport. Structural intermediates formed during the transition leak water molecules and may correlate to the uncoupled Cl⁻ion conductance observed experimentally in both prokaryotic and mammalian glutamate transporters. Mechanistic insights obtained from our study provide a structural framework for better development of therapeutic for neurological disorders. 

Archaeal group II chaperonins, also known as heat shock proteins (HSPs), are abundantly expressed in Sulfolobales. HSPα and HSPβ gene expression is upregulated during thermal shock. HSPs form large 18-mer complexes that assist in folding nascent proteins and protecting resident proteins during thermal stress. Engineered HSPs have been designed for industrial applications. Since temperature flux in the geothermal habitats of Sulfolobales impacts intracellular temperature, it follows that HSPs have developed thermotolerance. However, despite the low pH (i.e., pH < 4) typical for these habitats, intracellular pH in Sulfolobales is maintained at ~6.5. Therefore, it is not presumed that HSPs have evolved acid-tolerance. To test tolerance to low pH, HSPs were studied at various pH and temperature values. Both circular dichroism and intrinsic fluorescence indicate that HSPα and HSPβ retain structural integrity at neutral pH over a wide range of temperatures. Structural integrity is compromised for all HSPs at ultra-low pH (e.g., pH 2). Secondary structures in HSPs are resilient under mildly acidic conditions (pH 4) but Anilino naphthalene 8-sulfonate binding shows shifts in tertiary structure at lower pH. Trypsin digestion shows that the HSPβ-coh backbone is the most flexible and HSPβ is the most resilient. Overall, results suggest that HSPα and HSPβ exhibit greater thermostability than HSPβ-coh and that there are limits to HSP acid-tolerance. Molecular dynamics (MD) simulations complement the wet lab data. Specifically, MD suggests that the HSPβ secondary structure is the most stable. Also, despite similarities in pH- and temperature-dependent behavior, there are clear differences in how each HSP subtype is perturbed.