DEERefiner-assisted structural refinement using pulsed dipolar spectroscopy: a study on multidrug transporter LmrP.

Pulsed dipolar spectroscopy, such as double electron-electron resonance (DEER), has been underutilized in protein structure determination, despite its ability to provide valuable spatial information. In this study, we present DEERefiner, a user-friendly MATLAB-based GUI program that enables the modeling of protein structures by combining an initial structure and DEER distance restraints. We illustrate the effectiveness of DEERefiner by successfully modeling the ligand-dependent conformational changes of the proton-drug antiporter LmrP to an extracellular-open-like conformation with an impressive precision of 0.76 Å. Additionally, DEERefiner was able to uncover a previously hypothesized but experimentally unresolved proton-dependent conformation of LmrP, characterized as an extracellular-closed/partially intracellular-open conformation, with a precision of 1.16 Å. Our work not only highlights the ability of DEER spectroscopy to model protein structures but also reveals the potential of DEERefiner to advance the field by providing an accessible and applicable tool for precise protein structure modeling, thereby paving the way for deeper insights into protein function.

Structure and regulation of the BsYetJ calcium channel in lipid nanodiscs.

BsYetJ is a bacterial homolog of transmembrane BAX inhibitor-1 motif-containing 6 (TMBIM6) membrane protein that plays a key role in the control of calcium homeostasis. However, the BsYetJ (or TMBIM6) structure embedded in a lipid bilayer is uncharacterized, let alone the molecular mechanism of the calcium transport activity. Herein, we report structures of BsYetJ in lipid nanodiscs identified by double electron-electron resonance spectroscopy. Our results reveal that BsYetJ in lipid nanodiscs is structurally different from those crystallized in detergents. We show that BsYetJ conformation is pH-sensitive in apo state (lacking calcium), whereas in a calcium-containing solution it is stuck in an intermediate, inert to pH changes. Only when the transmembrane calcium gradient is established can the calcium-release activity of holo-BsYetJ occur and be mediated by pH-dependent conformational changes, suggesting a dual gating mechanism. Conformational substates involved in the process and a key residue D171 relevant to the gating of calcium are identified. Our study suggests that BsYetJ/TMBIM6 is a pH-dependent, voltage-gated calcium channel.

Mixed-Valence CuI /CuIII Metal-Organic Frameworks with Non-innocent Ligand for Multielectron Transfer.

We report two novel three-dimensional copper-benzoquinoid metal-organic frameworks (MOFs), [Cu4 L3 ]n and [Cu4 L3 ⋅ Cu(iq)3 ]n (LH4 =1,4-dicyano-2,3,5,6-tetrahydroxybenzene, iq=isoquinoline). Spectroscopic techniques and computational studies reveal the unprecedented mixed valency in MOFs, formal Cu(I)/Cu(III). This is the first time that formally Cu(III) species are witnessed in metal-organic extended solids. The coordination between the mixed-valence metal and redox-non-innocent ligand L, which promotes through-bond charge transfer between Cu metal sites, allows better metal-ligand orbital overlap of the d-π conjugation, leading to strong long-range delocalization and semiconducting behavior. Our findings highlight the significance of the unique mixed valency between formal Cu(I) and highly-covalent Cu(III), non-innocent ligand, and pore environments of these bench stable Cu(III)-containing frameworks on multielectron transfer and electrochemical properties.

Nanodisc Lipids Exhibit Singular Behaviors Implying Critical Phenomena.

Nanodiscs are broadly used for characterization of membrane proteins as they are generally assumed to provide a near-native environment. In fact, it is an open question whether the physical properties of lipids in nanodiscs and membrane vesicles of the same lipid composition are identical. Here, we investigate the properties of lipids (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dilauroyl-sn-glycero-3-phosphocholine, and their mixtures) in two different sample types, nanodiscs and multilamellar vesicles, by means of spin-label electron spin resonance techniques. Our results provide a quantitative description of lipid dynamics and ordering, elucidating the molecular details of how lipids in the two sample types behave differently in response to temperature and lipid composition. We show that the properties of lipids are altered in nanodiscs such that the dissimilarity of the fluid and gel lipid phases is reduced, and the first-order phase transitions are largely abolished in nanodiscs. We unveil that the ensemble of lipids in the middle of a nanodisc bilayer, as probed by the end-chain spin-label 16-PC, is promoted to a state close to a miscibility critical point, thereby rendering the phase transitions continuous. Critical phenomena have recently been proposed to explain features of the heterogeneity in native cell membranes. Our results lay the groundwork for how to establish a near-native environment in nanodiscs with simple organization of lipid components.

Bisphenol A induced apoptosis via oxidative stress generation involved Nrf2/HO-1 pathway and mitochondrial dependent pathways in human retinal pigment epithelium (ARPE-19) cells.

Bisphenol A (BPA) is an estrogen-like compound, and an environmental hormone, that is commonly used in daily life. Therefore, it may enter the human body through food or direct contact, causing BPA residues in blood and urine. Because most studies focused on the analysis of BPA in reproductive cells or tissues, regarding evidence the effect of BPA on human retinal pigment epithelium (ARPE-19) cells unavailable. Accordingly, the present study explored the cytotoxicity of BPA on ARPE-19 cells. After BPA treatment, the expression of Bcl-XL an antiapoptotic protein, in the mitochondria decreased, and the expression of Bax, a proapoptotic protein increased. Then the mitochondrial membrane potential was affected. BPA changed in mitochondrial membrane potential led to the release of cytochrome C, which activated caspase-9 to promote downstream caspase-3 leading to cytotoxicity. The nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and heme oxygenase 1 (HO-1) pathway play a major role in age-related macular degeneration. Our results showed that expression of HO-1 and Nrf2 suppressed by BPA. Superoxide dismutase and catalase, which Nrf2 downstream antioxidants, were degraded by BPA. AMP-activated kinase (AMPK), which can regulate the phosphorylation of Nrf2, and the phosphorylation of AMPK expression was reduced by BPA. Finally, BPA-induced ROS generation and cytotoxicity were reduced by N-acetyl-l-cysteine. Taken together, these results suggest that BPA induced ARPE-19 cells via oxidative stress, which was associated with down regulated Nrf2/HO-1 pathway, and the mitochondria dependent apoptotic signaling pathway.

Advanced wearable biosensors for the detection of body fluids and exhaled breath by graphene.

Given the huge economic burden caused by chronic and acute diseases on human beings, it is an urgent requirement of a cost-effective diagnosis and monitoring process to treat and cure the disease in their preliminary stage to avoid severe complications. Wearable biosensors have been developed by using numerous materials for non-invasive, wireless, and consistent human health monitoring. Graphene, a 2D nanomaterial, has received considerable attention for the development of wearable biosensors due to its outstanding physical, chemical, and structural properties. Moreover, the extremely flexible, foldable, and biocompatible nature of graphene provide a wide scope for developing wearable biosensor devices. Therefore, graphene and its derivatives could be trending materials to fabricate wearable biosensor devices for remote human health management in the near future. Various biofluids and exhaled breath contain many relevant biomarkers which can be exploited by wearable biosensors non-invasively to identify diseases. In this article, we have discussed various methodologies and strategies for synthesizing and pattering graphene. Furthermore, general sensing mechanism of biosensors, and graphene-based biosensing devices for tear, sweat, interstitial fluid (ISF), saliva, and exhaled breath have also been explored and discussed thoroughly. Finally, current challenges and future prospective of graphene-based wearable biosensors have been evaluated with conclusion. Graphene is a promising 2D material for the development of wearable sensors. Various biofluids (sweat, tears, saliva and ISF) and exhaled breath contains many relevant biomarkers which facilitate in identify diseases. Biosensor is made up of biological recognition element such as enzyme, antibody, nucleic acid, hormone, organelle, or complete cell and physical (transducer, amplifier), provide fast response without causing organ harm.

Genotoxic effects of 1-nitropyrene in macrophages are mediated through a p53-dependent pathway involving cytochrome c release, caspase activation, and PARP-1 cleavage.

Genotoxic stress from environmental pollutants plays a critical role in cytotoxicity. The most abundant nitro-polycyclic aromatic hydrocarbon in environmental pollutants, 1-nitropyrene (1-NP), is generated during fossil fuel, diesel, and biomass combustion under sunlight. Macrophages, the key regulators of the innate immune system, provide the first line of defense against pathogens. The toxic effects of 1-NP on macrophages remain unclear. Through a lactate dehydrogenase assay, we measured the cytotoxicity induced by 1-NP. Our results revealed that 1-NP induced genotoxicity also named DNA damage, including micronucleus formation and DNA strand breaks, in a concentration-dependent manner. Furthermore, 1-NP induced p53 phosphorylation and nuclear accumulation; mitochondrial cytochrome c release; caspase-3 and -9 activation and cleavage; and poly (ADP-ribose) polymerase-1 (PARP-1) cleavage in a concentration-dependent manner. Pretreatment with the PARP inhibitor, 3-aminobenzamide, significantly reduced cytotoxicity, genotoxicity, and PARP-1 cleavage induced by 1-NP. Pretreatment with the caspase-3 inhibitor, z-DEVD-fmk, significantly reduced cytotoxicity, genotoxicity, PARP-1 cleavage, and caspase 3 activation induced by 1-NP. Pretreatment with the p53 inhibitor, pifithrin-α, significantly reduced cytotoxicity, genotoxicity, PARP-1 cleavage, caspase 3 activation, and p53 phosphorylation induced by 1-NP. We propose that cytotoxicity and genotoxicity induced by 1-NP by PARP-1 cleavage via caspase-3 and -9 activation through cytochrome c release from mitochondria and its upstream p53-dependent pathway in macrophages.

Ligand-Based Reactivity of Oxygenation and Alkylation in Cobalt Complexes Binding with (Thiolato)phosphine Derivatives.

In our efforts to understand the nature of metal thiolates, we have explored the chemistry of cobalt ion supported by (thiolato)phosphine ligand derivatives. Herein, we synthesized and characterized a square-planar CoII complex binding with a bidentate (thiolato)phosphine ligand, Co(PS1″)2 (1) ([PS1″]- = [P(Ph)2(C6H3-3-SiMe3-2-S)]-). The complex activates O2 to form a ligand-based oxygenation product, Co(OPS1″)2 (2) ([OPS1″]- = [PO(Ph)2(C6H3-3-SiMe3-2-S)]-). In addition, an octahedral CoIII complex with a tridentate bis(thiolato)phosphine ligand, [NEt4][Co(PS2*)2] (3) ([PS2*]2- = [P(Ph)(C6H3-3-Ph-2-S)2]2-), was obtained. Compound 3 cleaves the C-Cl bond in dichloromethane via an S-based nucleophilic attack to generate a chloromethyl thioether group. Two isomeric products, [Co(PS2*)(PSSCH2Cl*)] (4 and 4') ([PSSCH2Cl*]- = [P(Ph)(C6H3-3-Ph-2-S)(C6H3-3-Ph-2-SCH2Cl)]-), were isolated and fully characterized. Both transformations, oxygenation of a CoII-bound phosphine donor in 1 and alkylation of a CoIII-bound thiolate in 3, were monitored by spectroscopic methods. These reaction products were isolated and fully characterized. Density functional theory (DFT, the B3LYP functional) calculations were performed to understand the electronic structure of 1 as well as the pathway of its transformation to 2.

{Fe(NO)(2)(9) Dinitrosyl Iron Complex Acting as a Vehicle for the NO Radical.

To carry and deliver nitric oxide with a controlled redox state and rate is crucial for its pharmaceutical/medicinal applications. In this study, the capability of cationic {Fe(NO)2}9 dinitrosyl iron complexes (DNICs) [(RDDB)Fe(NO)2]+ (R = Me, Et, Iso; RDDB = N,N'-bis(2,6-dialkylphenyl)-1,4-diaza-2,3-dimethyl-1,3-butadiene) carrying nearly unperturbed nitric oxide radical to form [(RDDB)Fe(NO)2(•NO)]+ was demonstrated and characterized by IR, UV-vis, EPR, NMR, and single-crystal X-ray diffractions. The unique triplet ground state of [(RDDB)Fe(NO)2(•NO)]+ results from the ferromagnetic coupling between two strictly orthogonal orbitals, one from Fe dz2 and the other a π*op orbital of a unique bent axial NO ligand, which is responsible for the growth of a half-field transition (ΔMS = 2) from 70 to 4 K in variable-temperature EPR measurements. Consistent with the NO radical character of coordinated axial NO ligand in complex [(MeDDB)Fe(NO)2(•NO)]+, the simple addition of MeCN/H2O into CH2Cl2 solution of complexes [(RDDB)Fe(NO)2(•NO)]+ at 25 °C released NO as a neutral radical, as demonstrated by the formation of [S5Fe(NO)2]- from [S5Fe(µ-S)2FeS5]2-.

The role of conformational heterogeneity in regulating the apoptotic activity of BAX protein.

While activation of BAX is required for initiating mitochondria-mediated apoptosis, the underlying mechanisms remain unsettled. We studied conformations of BAX protein using pressure- and temperature-resolved ESR techniques and obtained the thermodynamic properties of the conformations. We show that inactive BAX is structurally heterogeneous and exists in equilibrium between two major populations of the conformations, UM and UM', of which the former is thermodynamically favored at room temperature. An increase in the population of UM', induced by either pressure or point mutations of BAX, renders BAX susceptible to oligomerization, which leads to cell death. This study uncovers the biological significance of BAX conformations and shows that the pro-apoptotic activity of BAX can be triggered by altering the equilibrium between the two states. It suggests that therapeutic intervention may focus on shifting the balance in the conformational heterogeneity.

Oligomerization process of Bcl-2 associated X protein revealed from intermediate structures in solution.

Upon apoptotic stress, Bcl-2 associated X (BAX) protein undergoes conformational changes and oligomerizes, leading to the mitochondrial membrane permeabilization and cell death. While structures of the resultant oligomer have been extensively studied, little is known about the intermediates that describe the reaction pathway from the inactive monomers to activated oligomers. Here we characterize the intermediate structures of BAX using combined small-angle X-ray scattering (SAXS) with on-line gel-filtration and electron spin resonance (ESR). The intermediates, including monomers, dimers, and tetramers, are reconstructed via integrating the SAXS-envelopes and ESR-determined skeleton structures. The hence revealed structures suggest a linear oligomerization of BAX utilizing the extended dimers with the two flexible α6 chains protruded out as ditopic ligands. The results of molecular dynamics simulation also support the ditopic dimer conformation with mobile α6. The ditopic dimers could further wind into a helical rod structure with three dimers in one helical turn. Our results not only reveal the on-pathway intermediates, but also suggest a ditopic oligomerization mechanism that may bridge the observed intermediate structures in solution to the large BAX assemblies lately observed on mitochondria.

A Structurally Characterized Nonheme Cobalt-Hydroperoxo Complex Derived from Its Superoxo Intermediate via Hydrogen Atom Abstraction.

Bubbling O2 into a THF solution of CoII(BDPP) (1) at -90 °C generates an O2 adduct, Co(BDPP)(O2) (3). The resonance Raman and EPR investigations reveal that 3 contains a low spin cobalt(III) ion bound to a superoxo ligand. Significantly, at -90 °C, 3 can react with 2,2,6,6-tetramethyl-1-hydroxypiperidine (TEMPOH) to form a structurally characterized cobalt(III)-hydroperoxo complex, CoIII(BDPP)(OOH) (4) and TEMPO•. Our findings show that cobalt(III)-superoxo species are capable of performing hydrogen atom abstraction processes. Such a stepwise O2-activating process helps to rationalize cobalt-catalyzed aerobic oxidations and sheds light on the possible mechanism of action for Co-bleomycin.

Highly Facet-Dependent Photocatalytic Properties of Cu2 O Crystals Established through the Formation of Au-Decorated Cu2 O Heterostructures.

This work confirms the presence of a large facet-dependent photocatalytic activity of Cu2 O crystals through sparse deposition of gold particles on Cu2 O cubes, octahedra, and rhombic dodecahedra. Au-decorated Cu2 O rhombic dodecahedra and octahedra showed greatly enhanced photodegradation rates of methyl orange resulting from a better separation of the photogenerated electrons and holes, with the rhombic dodecahedra giving the best efficiency. Au-Cu2 O core-shell rhombic dodecahedra also displayed a better photocatalytic activity than pristine rhombic dodecahedra. However, Au-deposited Cu2 O cubes, pristine cubes, and Au-deposited small nanocubes bound by entirely {100} facets are all photocatalytically inactive. X-ray photoelectron spectra (XPS) showed identical copper peak positions for these Au-decorated crystals. Remarkably, electron paramagnetic resonance (EPR) measurements indicated a higher production of hydroxyl radicals for the photoirradiated Cu2 O rhombic dodecahedra than for the octahedra, but no radicals were produced from photoirradiated Cu2 O cubes. The Cu2 O {100} face may present a high energy barrier through its large band edge bending and/or electrostatic repulsion, preventing charge carriers from reaching to this surface. The conventional photocatalysis model fails in this case. The facet-dependent photocatalytic differences should be observable in other semiconductor systems whenever a photoinduced charge-transfer process occurs across an interface.

Crystal Structure Analysis of the Repair of Iron Centers Protein YtfE and Its Interaction with NO.

Molecular mechanisms underlying the repair of nitrosylated [Fe-S] clusters by the microbial protein YtfE remain poorly understood. The X-ray crystal structure of YtfE, in combination with EPR, magnetic circular dichroism (MCD), UV, and (17) O-labeling electron spin echo envelope modulation measurements, show that each iron of the oxo-bridged Fe(II) -Fe(III) diiron core is coordinatively unsaturated with each iron bound to two bridging carboxylates and two terminal histidines in addition to an oxo-bridge. Structural analysis reveals that there are two solvent-accessible tunnels, both of which converge to the diiron center and are critical for capturing substrates. The reactivity of the reduced-form Fe(II) -Fe(II) YtfE toward nitric oxide demonstrates that the prerequisite for N2 O production requires the two iron sites to be nitrosylated simultaneously. Specifically, the nitrosylation of the two iron sites prior to their reductive coupling to produce N2 O is cooperative. This result suggests that, in addition to any repair of iron centers (RIC) activity, YtfE acts as an NO-trapping scavenger to promote the NO to N2 O transformation under low NO flux, which precedes nitrosative stress.

NMR characterization of the electrostatic interaction of the basic residues in HDGF and FGF2 during heparin binding.

Electrostatic interaction is a major driving force in the binding of proteins to highly acidic glycosaminoglycan, such as heparin. Although NMR backbone chemical shifts have generally been used to identify the heparin-binding site on a protein, however, there is no correlation between the binding free energies and the perturbed backbone chemical shifts for individual residues. The binding event occurs at the end of a side chain of basic residue, and does not require causing significant alterations in the backbone environment at a distance of multiple bonds. We used the H2CN NMR pulse sequence to detect heparin binding through the side-chain resonances Hε-Cε-Nζ of Lys and Hδ-Cδ-Nε of Arg in the two proteins of hepatoma-derived growth factor (HDGF) and basic fibroblast growth factor (FGF2). H2CN titration experiments revealed chemical shift perturbations in the side chains, which were correlated with the free energy changes in various mutants. The residues K19 in HDGF and K125 in FGF2 demonstrated the most significant perturbations, consistent with our previous observation that the two residues are crucial for binding. The result suggests that H2CN NMR provides a precise evaluation for the electrostatic interactions. The discrepancy observed between backbone and side chain chemical shifts is correlated to the solvent accessibility of residues that the K19 and K125 backbones are highly buried with the restricted backbone conformation and are not strongly affected by the events at the end of the side chains.

Mesopores provide an amorphous state suitable for studying biomolecular structures at cryogenic temperatures.

In nano-confinements, aqueous solutions can be found to remain in a liquid state at subfreezing temperatures. The finding provides a means of entering into previously inaccessible temperature regions for studying the dynamics and structure of bulk liquid. Here we show that studying biomolecular structures in nano-confinements improves the accuracy of cryostructures and provides better insight into the relationship between hydration water and biomolecules. Synthetic prion protein peptides are studied in two experimental conditions: (i) in confined nanochannels within mesoporous materials, and (ii) in vitrified bulk solvents, with a temperature range of 50-275 K, using cw/pulse ESR techniques. A large inhomogeneous lineshape broadening is only observed for the spectra from the vitrified bulk solvent below 70 K, suggesting a possible peptide clustering in the solution. The spin-counting and distance measurements by DEER-ESR provide further evidence that peptides are dispersed homogeneously in mesopores but heterogeneously in vitrified solvents wherein the biomolecular structure is disturbed due to heterogeneity in the bulk solvent structure. Our study demonstrates that the nanospace within mesoporous materials provides an amorphous environment that is better than vitrified bulk solvent for studying biostructures at cryogenic temperatures.

Manipulating the Rate and Overpotential for Electrochemical Water Oxidation: Mechanistic Insights for Cobalt Catalysts Bearing Noninnocent Bis(benzimidazole)pyrazolide Ligands.

Electrochemical water oxidation is known as the anodic reaction of water splitting. Efficient design and earth-abundant electrocatalysts are crucial to this process. Herein, we report a family of catalysts (1-3) bearing bis(benzimidazole)pyrazolide ligands (H 2 L1-H 2 L3). H 2 L3 contains electron-donating substituents and noninnocent components, resulting in catalyst 3 exhibiting unique performance. Kinetic studies show first-order kinetic dependence on [3] and [H2O] under neutral and alkaline conditions. In contrast to previously reported catalyst 1, catalyst 3 exhibits an insignificant kinetic isotope effect of 1.25 and zero-order dependence on [NaOH]. Based on various spectroscopic methods and computational findings, the L3Co2 III(µ-OH) species is proposed to be the catalyst resting state and the nucleophilic attack of water on this species is identified as the turnover-limiting step of the catalytic reaction. Computational studies provided insights into how the interplay between the electronic effect and ligand noninnocence results in catalyst 3 acting via a different reaction mechanism. The variation in the turnover-limiting step and catalytic potentials of species 1-3 leads to their catalytic rates being independent of the overpotential, as evidenced by Eyring analysis. Overall, we demonstrate how ligand design may be utilized to retain good water oxidation activity at low overpotentials.

Comprehensive characterization of polyproline tri-helix macrocyclic nanoscaffolds for predictive ligand positioning.

Multivalent ligands hold promise for enhancing avidity and selectivity to simultaneously target multimeric proteins, as well as potentially modulating receptor signaling in pharmaceutical applications. Essential for these manipulations are nanosized scaffolds that precisely control ligand display patterns, which can be achieved by using polyproline oligo-helix macrocyclic nanoscaffolds via selective binding to protein oligomers and cell surface receptors. This work focuses on synthesis and structural characterization of different-sized polyproline tri-helix macrocyclic (PP3M) scaffolds. Through combined analysis of circular dichroism (CD), small- and wide-angle X-ray scattering (SWAXS), electron spin resonance (ESR) spectroscopy, and molecular modeling, a non-coplanar tri-helix loop structure with partially crossover helix ends is elucidated. This structural model aligns well with scanning tunneling microscopy (STM) imaging. The present work enhances the precision of nanoscale organic synthesis, offering prospects for controlled ligand positioning on scaffolds. This advancement paves the way for further applications in nanomedicine through selective protein interaction, manipulation of cell surface receptor functions, and developments of more complex polyproline-based nanostructures.

Concurrent observation of bulk and protein hydration water by spin-label ESR under nanoconfinement.

Under nanoconfinement the formation of crystalline ice is suppressed, allowing the study of water dynamics at subfreezing temperatures. Here we report a temperature-dependent investigation (170-260 K) of the behavior of hydration water under nanoconfinement by ESR techniques. A 26-mer-long peptide and the Bax protein are studied. This study provides site-specific information about the different local hydrations concurrently present in the protein/peptide solution, enabling a decent comparison of the hydration molecules-those that are buried inside, in contact with, and detached from the protein surface. Such a comparison is not possible without employing ESR under nanoconfinement. Though the confined bulk and surface hydrations behave differently, they both possess a transition similar to the reported fragile-to-strong crossover transition around 220 K. On the contrary, this transition is absent for the hydration near the buried sites of the protein. The activation energy determined under nanoconfinement is found to be lower in surface hydration than in bulk hydration. The protein structural flexibility, derived from the interspin distance distributions P(r) at different temperatures, is obtained by dipolar ESR spectroscopy. The P(r) result demonstrates that the structural flexibility is strongly correlated with the transition in the surface water, corroborating the origin of the protein dynamical transition at subfreezing temperatures.

A gating mechanism of the BsYetJ calcium channel revealed in an endoplasmic reticulum lipid environment.

The transmembrane BAX inhibitor-1-containing motif 6 (TMBIM6) is suggested to modulate apoptosis by regulating calcium homeostasis in the endoplasmic reticulum (ER). However, the precise molecular mechanism underlying this calcium regulation remains poorly understood. To shed light on this issue, we investigated all negatively charged residues in BsYetJ, a bacterial homolog of TMBIM6, using mutagenesis and fluorescence-based functional assays. We reconstituted BsYetJ in membrane vesicles with a lipid composition similar to that of the ER. Our results show that the charged residues E49 and R205 work together as a major gate, regulating calcium conductance in these ER-like lipid vesicles. However, these residues become largely inactive when reconstituted in other lipid environments. In addition, we found that D195 acts as a minor filter compared to the E49-R205 dyad. Our study uncovers a previously unknown function of BsYetJ/TMBIM6 in the calcium-dependent inactivation of BsYetJ, providing a framework for the development of a lipid-dependent mechanistic model of BsYetJ that will facilitate our understanding of calcium-dependent apoptosis.