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.

Structural Insights into the Binding and Degradation Mechanisms of Protoporphyrin IX by the Translocator Protein TSPO.

The 18 kDa translocator protein (TSPO) has gained considerable attention as a clinical biomarker for neuroinflammation and a potential therapeutic target. However, the mechanisms by which TSPO associates with ligands, particularly the endogenous porphyrin ligand protoporphyrin IX (PpIX), remain poorly understood. In this study, we employed mutagenesis- and spectroscopy-based functional assays to investigate TSPO-mediated photo-oxidative degradation of PpIX and identify key residues involved in the reaction. We provide structural evidence using electron spin resonance, which sheds light on the highly conserved intracellular loop (LP1) connecting transmembrane 1 (TM1) and TM2. Our findings show that LP1 does not act as a lid to regulate ligand binding; instead, it interacts strongly with the TM3-TM4 linker (LP3) to stabilize the local structure of LP3. This LP1-LP3 interaction is crucial for maintaining the binding pocket structure, which is essential for proper ligand binding. Our results also demonstrate that PpIX accesses the pocket through the lipid bilayer without requiring conformational changes in TSPO. This study provides an improved understanding of TSPO-mediated PpIX degradation, highlighting potential therapeutic strategies to regulate the reaction.

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.

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.

Cholesterol twists the transmembrane Di-Gly region of amyloid-precursor protein.

Nearly 95% of Alzheimer's disease (AD) occurs sporadically without genetic linkage. Aging, hypertension, high cholesterol content, and diabetes are known nongenomic risk factors of AD. Aggregation of Aß peptides is an initial event of AD pathogenesis. Aß peptides are catabolic products of a type I membrane protein called amyloid precursor protein (APP). Aß40 is the major product, whereas the 2-residue-longer version, Aß42, induces amyloid plaque formation in the AD brain. Since cholesterol content is one risk factor for sporadic AD, we aimed to explore whether cholesterol in the membrane affects the structure of the APP transmembrane region, thereby modulating the γ-secretase cutting behavior. Here, we synthesized several peptides containing the APP transmembrane region (sequence 693-726, corresponding to the Aß22-55 sequence) with one or two Cys mutations for spin labeling. We performed three electron spin resonance experiments to examine the structural changes of the peptides in liposomes composed of dioleoyl phosphatidylcholine and different cholesterol content. Our results show that cholesterol increases membrane thickness by 10% and peptide length accordingly. We identified that the di-glycine region of Aß36-40 (sequence VGGVV) exhibits the most profound change in response to cholesterol compared with other segments, explaining how the presence of cholesterol affects the γ-secretase cutting site. This study provides spectroscopic evidence showing how cholesterol modulates the structure of the APP transmembrane region in a lipid bilayer.

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.

Nitrosoarenes Implement Cascade Cyclization of 1-Allenyl-2-alkynylbenzenes through Diradical Mechanism.

This work reports cascade cyclization between 1-allenyl-2-alkynylbenzenes and nitrosoarenes. When these two components reacted alone under N2, N,O-functionalized indane-fused isoxazolidines 3 were obtained selectively. DFT calculations verify that this reaction sequence involves unprecedented nitrone/alkyne cycloadditions, followed by diradical rearrangement.

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.

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.

Location of the cross-ß structure in prion fibrils: A search by seeding and electron spin resonance spectroscopy.

Prion diseases are transmissible fatal neurodegenerative disorders spreading between humans and other mammals. The pathogenic agent, prion, is a protease-resistant, ß-sheet-rich protein aggregate, converted from a membrane protein called PrPC . PrPSc is the misfolded form of PrPC and undergoes self-propagation to form the infectious amyloids. Since the key hallmark of prion disease is amyloid formation, identifying and studying which segments are involved in the amyloid core can provide molecular details about prion diseases. It has been known that the prion protein could also form non-infectious fibrils in the presence of denaturants. In this study, we employed a combination of site-directed nitroxide spin-labeling, fibril seeding, and electron spin resonance (ESR) spectroscopy to identify the structure of the in vitro-prepared full-length mouse prion fibrils. It is shown that in the in vitro amyloidogenesis, the formation of the amyloid core is linked to an α-to-ß structural transformation involving the segment 160-224, which contains strand 2, helix 2, and helix 3. This method is particularly suitable for examining the hetero-seeded amyloid fibril structure, as the unlabeled seeds are invisible by ESR spectroscopy. It can be applied to study the structures of different strains of infectious prions or other amyloid fibrils in the future.

Fine Particulate Matter Exposure Levels in Patients with Normal-Tension Glaucoma and Primary Open-Angle Glaucoma: A Population-Based Study from Taiwan.

Patients with NTG or POAG with more than one outpatient or discharge diagnosis from the ophthalmology department were included in the study. These data were merged with the PM2.5 data from the Air Quality Monitoring Network for analysis. This was a case−control study, with 1006 participants in the NTG group and 2533 in the POAG group. To investigate fine particulate matter (PM2.5) exposure levels in patients with normal-tension glaucoma (NTG) and primary open-angle glaucoma (POAG), patient data were obtained from Taiwan's Longitudinal Health Insurance Database 2000 for the 2008 to 2013 period. We used a multivariate logic regression model to assess the risk for each participant. The PM2.5 exposure levels were divided into four groups: <25th percentile (Q1), <617 µg/mm3; 25th to 50th percentile (Q2), 617 to 1297 µg/mm3; 50th to 75th percentile (Q3), 1297 to 2113 µg/mm3; and >75th percentile (Q4), >2113 µg/mm3. The results are expressed in terms of odds ratio (OR) and 95% CI. A multiple logistic regression was used to compare the results of the NTG group with those of the POAG group. Compared with the PM2.5 Q1 level, the OR of the PM2.5 Q2 level was 1.009 (95% CI 0.812−1.254), the PM2.5 Q3 level was 1.241 (95% CI 1.241−1.537, p < 0.05), and the PM2.5 Q4 level was 1.246 (95% CI 1.008−1.539, p < 0.05). Our research reveals that compared with POAG, the risk of developing NTG is more closely related with PM2.5 exposure, and PM2.5 has a concentration−dose effect. It is hoped that in the future, in the clinical judgment of NTG and POAG, the level of PM2.5 in the environment can be taken as a risk factor.

Simple Cryoprotectant-Free Method to Advance Pulsed Dipolar ESR Spectroscopy for Capturing Protein Conformational Ensembles.

Double electron-electron resonance (DEER) is a powerful technique for studying protein conformations. To preserve the room-temperature ensemble, proteins are usually shock-frozen in liquid nitrogen prior to DEER measurements. The use of cryoprotectant additives is, therefore, necessary to ensure the formation of a vitrified state. Here, we present a simple modification of the freezing process using a flexible fused silica microcapillary, which increases the freezing rates and thus enables DEER measurement without the use of cryoprotectants. The Bid protein, which is highly sensitive to cryoprotectant additives, is used as a model. We show that DEER with the simple modification can successfully reveal the cold denaturation of Bid, which was not possible with the conventional DEER preparations. The DEER result reveals the nature of Bid folding. Our method advances DEER for capturing the chemically and thermally induced conformational changes of a protein in a cryoprotectant-free medium.

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.

Air Pollutant Particles, PM2.5, Exposure and Glaucoma in Patients with Diabetes: A National Population-Based Nested Case-Control Study.

The global prevalence of diabetes mellitus (DM) has reached 20%. Air pollutants with a particle size of less than 2.5 µm (PM2.5) are a globally recognized risk factor for diabetes and glaucoma. We examined whether the risk of glaucoma would decrease or increase when patients with DM were exposed to different PM2.5 concentrations. Data were obtained from the National Health Insurance Research Database (NHIRD) of Taiwan and the Air Quality Monitoring Network between 2008 and 2013. This nested case-control study involved 197 DM patients with glaucoma and 788 DM patients without glaucoma. Cases and controls were matched (1:4) by gender, age (±5 years), and index date (±6 months), and their data were entered in a logistic regression model adjusted for gender, age, urbanization level, income level, and comorbidities. The odds ratio (OR) of glaucoma at PM2.5 exposure concentration in the fourth quartile (Q4) compared with in the first quartile (Q1) was 1.7 (95% CI: 1.084-2.764). For glaucoma risk, the OR was 1.013 (95% CI: 1.006-1.020) at a PM2.5 exposure concentration in Q1, 1.004 (95% CI: 1.001-1.007) in the third quartile (Q3), and 1.003 (95% CI: 1.001-1.004) in Q4. In the subgroup analysis of patients living in non-emerging towns and non-agricultural towns, the OR for glaucoma in Q4 compared with in Q1 was 2.1 (95% CI: 1.229-3.406) and 1.8 (95% CI: 1.091-2.803), respectively (p trend = 0.001 and 0.011). For patients without migraine, the OR for glaucoma was 1.7 (95% CI: 1.074-2.782; p = 0.006). These results demonstrate that, for patients with DM, PM2.5 increased the risk of glaucoma and PM2.5 was an independent risk factor for glaucoma in patients with DM.

Protein Stability Depends Critically on the Surface Hydrogen-Bonding Network: A Case Study of Bid Protein.

Understanding how proteins retain structural stability is not only of fundamental importance in biophysics but also critical to industrial production of antibodies and vaccines. Protein stability is known to depend mainly on two effects: internal hydrophobicity and H-bonding between the protein surface and solvent. A challenging task is to identify their individual contributions to a protein. Here, we investigate the structural stability of the apoptotic Bid protein in solutions containing various concentrations of guanidinium hydrochloride and urea using a combination of recently developed methods including the QTY (glutamine, threonine, and tyrosine) code and electron spin resonance-based peak-height analysis. We show that when the internal hydrophobicity of Bid is broken down using the QTY code, the surface H-bonding alone is sufficient to retain the structural stability intact. When the surface H-bonding is disrupted, Bid becomes sensitive to the temperature-dependent internal hydrophobicity such that it exhibits a reversible cold unfolding above water's freezing point. Using the combined approach, we show that the free-energy contributions of the two effects can be more reliably obtained. The surface H bonds are more important than the other effect in determining the structural stability of Bid protein.

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.

Association Between PM2.5 Exposure Level and Primary Open-Angle Glaucoma in Taiwanese Adults: A Nested Case-Control Study.

Primary open-angle glaucoma (POAG) is the most common type of glaucoma. However, little is known about POAG in adults and exposure to air pollution. The current study aims to investigate whether exposure to particulate matter with a mass median aerodynamic diameter of ≤2.5 µm (PM2.5) is associated with POAG diagnosis. Patient data were obtained from the Longitudinal Health Insurance Database 2010 (LHID2010) of Taiwan for the 2008-2013 period. PM2.5 concentration data, collected from the Ambient Air Quality Monitoring Network established by the Environmental Protection Administration of Taiwan, were categorized into four groups according to World Health Organization (WHO) exposure standards for PM2.5. We estimated the odds ratios (ORs) and 95% CIs for risk factors for POAG with logistic regression. The OR of per WHO standard level increase was 1.193 (95% CI 1.050-1.356). Compared with the normal level, the OR of WHO 2.0 level was 1.668 (95% CI 1.045-2.663, P < 0.05). After excluding confounding risk factors for POAG in this study, we determined that increased PM2.5 exposure is related to POAG risk (ORs > 1, P < 0.05). In this study, PM2.5 was an independent factor associated with open-angle glaucoma. Further research is required to better understand the mechanisms connecting PM2.5 and open-angle glaucoma.

Stepwise activation of the pro-apoptotic protein Bid at mitochondrial membranes.

Caspase-8-cleaved Bid (cBid) associates with mitochondria and promotes the activation of BAX, leading to mitochondria outer membrane permeabilization (MOMP) and apoptosis. However, current structural models of cBid are largely based on studies using membrane vesicles and detergent micelles. Here we employ spin-label ESR and site-directed PEGylation methods to identify conformations of cBid at real mitochondrial membranes, revealing stepwise mechanisms in the activation process. Upon the binding of cBid to mitochondria, its structure is reorganized to expose the BH3 domain while leaving the structural integrity only slightly altered. The mitochondria-bound cBid is in association with Mtch2 and it remains in the primed state until interacting with BAX. The interaction subsequently triggers the fragmentation of cBid, causes large conformational changes, and promotes BAX-mediated MOMP. Our results reveal structural differences of cBid between mitochondria and other lipid-like environments and, moreover, highlight the role of the membrane binding in modifying cBid structure and assisting the inactive-to-active transition in function.

Structural polymorphism and substrate promiscuity of a ribosome-associated molecular chaperone.

Trigger factor (TF) is a highly conserved multi-domain molecular chaperone that exerts its chaperone activity at the ribosomal tunnel exit from which newly synthesized nascent chains emerge. TF also displays promiscuous substrate binding for a large number of cytosolic proteins independent of ribosome binding. We asked how TF recognizes a variety of substrates while existing in a monomer-dimer equilibrium. Paramagnetic nuclear magnetic resonance (NMR) and electron spin resonance (ESR) spectroscopy were used to show that dimeric TF displays a high degree of structural polymorphism in solution. A series of peptides has been generated to quantify their TF binding affinities in relation with their sequence compositions. The results confirmed a previous predication that TF preferentially binds to peptide fragments that are rich in aromatic and positively charged amino acids. NMR paramagnetic relaxation enhancement analysis showed that TF utilizes multiple binding sites, located in the chaperone domain and part of the prolyl trans-cis isomerization domain, to interact with these peptides. Dimerization of TF effectively sequesters most of the substrate binding sites, which are expected to become accessible upon binding to the ribosome as a monomer. As TF lacks ATPase activity, which is commonly used to trigger conformational changes within molecular chaperones in action, the ribosome-binding-associated disassembly and conformational rearrangements may be the underlying regulatory mechanism of its chaperone activity.

Dissecting the Conformational Dynamics of the Bile Acid Transporter Homologue ASBTNM.

Apical sodium-dependent bile acid transporter (ASBT) catalyses uphill transport of bile acids using the electrochemical gradient of Na+ as the driving force. The crystal structures of two bacterial homologues ASBTNM and ASBTYf have previously been determined, with the former showing an inward-facing conformation, and the latter adopting an outward-facing conformation accomplished by the substitution of the critical Na+-binding residue glutamate-254 with an alanine residue. While the two crystal structures suggested an elevator-like movement to afford alternating access to the substrate binding site, the mechanistic role of Na+ and substrate in the conformational isomerization remains unclear. In this study, we utilized site-directed alkylation monitored by in-gel fluorescence (SDAF) to probe the solvent accessibility of the residues lining the substrate permeation pathway of ASBTNM under different Na+ and substrate conditions, and interpreted the conformational states inferred from the crystal structures. Unexpectedly, the crosslinking experiments demonstrated that ASBTNM is a monomer protein, unlike the other elevator-type transporters, usually forming a homodimer or a homotrimer. The conformational dynamics observed by the biochemical experiments were further validated using DEER measuring the distance between the spin-labelled pairs. Our results revealed that Na+ ions shift the conformational equilibrium of ASBTNM toward the inward-facing state thereby facilitating cytoplasmic uptake of substrate. The current findings provide a novel perspective on the conformational equilibrium of secondary active transporters.