Conformational Switch Research Articles

Constant-pH MD simulations of the protonation-triggered conformational switching in diphtheria toxin translocation domain

Protonation of key residues in the diphtheria toxin translocation (T)-domain triggered by endosomal acidification is critical for inducing a series of conformational transitions critical for the cellular entry of the toxin. Previous experiments revealed the importance of histidine residues in modulating pH-dependent transitions. They suggested the presence of a “safety latch” preventing premature refolding of the T-domain by a yet poorly understood mechanism. Here, we used constant-pH molecular dynamics simulations to systematically investigate the protonation sequence in the wild-type T-domain and the following mutants: H223Q, H257Q, E259Q, and H223Q/H257Q. Comparison of these computational results with previous experimental data on T-domain stability and activity with the H-to-Q replacements confirms the role of H223 (pKa = 6.5) in delaying the protonation of the main trigger, H257 (pKa = 2.2 in the WT and pKa = 4.9 in H223Q). Our calculations also reveal a very low pKa for a neighboring acidic residue E259, which does not get protonated even during simulations at pH 3. This residue also contributes to the formation of the safety latch, with the pKa of H257 increasing from 2.2 to 5.1 upon E259Q replacement. In contrast, the latter replacement has virtually no effect on the protonation of the H223. Thus, we conclude that the interplay of the protonation in the H223/H257/E259 triad has evolved to prevent triggering the accidental refolding of the T-domain by a fluctuation in the protonation of the main trigger at neutral pH, before the incorporation of the toxin inside the endosome. Subsequent acidification of the endosome overcomes the safety latch and triggers conformational switching via repulsion of H223+ and H257+. This protonation/conformation relationship corroborates experimental findings and offers a detailed stepwise molecular description of the transition mechanism, which can be instrumental in optimizing the potential applications of the T-domain for targeted delivery of therapies to tumors and other diseased acidic tissues.

The U1-70K and SRSF1 interaction is modulated by phosphorylation during the early stages of spliceosome assembly.

In eukaryotes, pre-mRNA splicing is vital for RNA processing and orchestrated by the spliceosome, whose assembly starts with the interaction between U1-70K and SR proteins. Despite the significance of the U1-70K/SR interaction, the dynamic nature of the complex and the challenges in obtaining soluble U1-70K have impeded a comprehensive understanding of the interaction at the structural level for decades. We overcome the U1-70K solubility issues, enabling us to characterize the interaction between U1-70K and SRSF1, a representative SR protein. We unveil specific interactions: phosphorylated SRSF1 RS with U1-70K BAD1, and SRSF1 RRM1 with U1-70K RRM. The RS/BAD1 interaction plays a dominant role, whereas the interaction between the RRM domains further enhances the stability of the U1-70K/SRSF1 complex. The RRM interaction involves the C-terminal extension of U1-70K RRM and the conserved acid patches on SRSF1 RRM1 that is involved in SRSF1 phase separation. Our circular dichroism spectra reveal that BAD1 adapts an α-helical conformation and RS is intrinsically disordered. Intriguingly, BAD1 undergoes a conformation switch from α-helix to β-strand and random coil upon RS binding. In addition to the regulatory mechanism via SRSF1 phosphorylation, the U1-70K/SRSF1 interaction is also regulated by U1-70K BAD1 phosphorylation. We find that U1-70K phosphorylation inhibits the U1-70K and SRSF1 interaction. Our structural findings are validated through in vitro splicing assays and in-cell saturated domain scanning using the CRISPR method, providing new insights into the intricate regulatory mechanisms of pre-mRNA splicing.

A bivariate fluorescence biosensor based on Janus DNA nanoarchitecture-loaded dual-emissive Ag nanoclusters as bi-responsive signaling reporters

Constructing label-free bivariate fluorescence biosensor would be intriguing and desired for the recognizable and accurate detection of two specific DNA segments, yet the design of functional DNA structures with low overlapped interference might be challenging. Herein in this work, a double-faced Janus DNA nanoarchitecture (JDNA) with bi-responsive recognition regions on opposite sides was assembled, which consisted of two substrate strands and two template strands for loading green-/red-emissive Ag nanoclusters (gAgNC and rAgNC) as bivariate signaling reporters. Of note, the hybridized double helix in the middle rationally oriented two flank faces and stabilized the rigid conformation of JDNA, while the template sequences of bicolor clusters were blocked to minimize non-specific background leakage. Upon inputting two targets, the discernible hairpins lost their hairpin structures due to forming two dsDNA complexes. They were executed to simultaneously invade JDNA for activating two individual target-recycled strand displacement (TRSD) events, guiding signal transduction and efficient amplification. Consequently, the clustering templates were unlocked via the tailored conformation switch of JDNA, in which gAgNC and rAgNC were in situ synthesized in two diagonal positions, thereby significantly emitting bi-responsive signal without cross interference. Benefited from the logic integration of double-faced JDNA and TRSD, a label-free, sensitive and specific bivariate fluorescence approach was developed, which would open a new avenue for the potential application in biosensing and bioanalysis.

An emerging assay for rapid diagnosis of live Salmonella Typhimurium by exploiting aqueous/liquid crystal interface

The rapid and accurate identification of live pathogens with high proliferative ability is in great demand to mitigate foodborne infection outbreaks. Herein, we have developed an ultrasensitive image-based aptasensing array to directly detect live Salmonella typhimurium (S.T) cells. This method relies on the long-range orientation of surfactant-decorated liquid crystals (LCs) and the superiority of aptamers (aptST). The self-assembling of hydrophobic surfactant tails leads to a perpendicular/vertical ordered film at the aqueous/LC interface and signal-off response. The addition of aptST perturbed LCs’ ordering into a planar/tilted state at the aqueous phase due to electrostatic interactions between the surfactant with the aptST, and a signal-on response. Following the conformational switch of aptST in the presence of live S. typhimurium, a relative reversing signal-off response was observed upon the target concentration. This aptasensor could promptly confirm the presence of S. typhimurium without intricate DNA-extraction or pre-enrichment stats over a linear range of 1–1.1 × 106 CFU/mL and a detection limit of 1.2 CFU/mL within ∼30 min. These results were successfully validated using molecular and culture-based methods in spiked-milk samples, with a 92.61–104.61 % recovery value. Meanwhile, the flexibility of this portable sensing platform allows for its development and adoption for the precise detection of various pathogens in food and the environment.

Peri active site catalysis of proline isomerisation is the molecular basis of allomorphy in β-phosphoglucomutase

Metabolic regulation occurs through precise control of enzyme activity. Allomorphy is a post-translational fine control mechanism where the catalytic rate is governed by a conformational switch that shifts the enzyme population between forms with different activities. β-Phosphoglucomutase (βPGM) uses allomorphy in the catalysis of isomerisation of β-glucose 1-phosphate to glucose 6-phosphate via β-glucose 1,6-bisphosphate. Herein, we describe structural and biophysical approaches to reveal its allomorphic regulatory mechanism. Binding of the full allomorphic activator β-glucose 1,6-bisphosphate stimulates enzyme closure, progressing through NAC I and NAC III conformers. Prior to phosphoryl transfer, loops positioned on the cap and core domains are brought into close proximity, modulating the environment of a key proline residue. Hence accelerated isomerisation, likely via a twisted anti/C4-endo transition state, leads to the rapid predominance of active cis-P βPGM. In contrast, binding of the partial allomorphic activator fructose 1,6-bisphosphate arrests βPGM at a NAC I conformation and phosphoryl transfer to both cis-P βPGM and trans-P βPGM occurs slowly. Thus, allomorphy allows a rapid response to changes in food supply while not otherwise impacting substantially on levels of important metabolites.

Activation of the influenza B M2 proton channel (BM2).

Influenza B viruses have co-circulated during most seasonal flu epidemics and can cause significant human morbidity and mortality due to their rapid mutation, emerging drug resistance, and severe impact on vulnerable populations. The influenza B M2 proton channel (BM2) plays an essential role in viral replication, but the mechanisms behind its symmetric proton conductance and the involvement of a second histidine (His27) cluster remain unclear. Here we perform the membrane- enabled continuous constant-pH molecular dynamics simulations on wildtype BM2 and a key H27A mutant to explore its pH-dependent conformational switch. Simulations capture the activation as the first histidine (His19) protonates and reveal the transition at lower pH values compared to AM2 is a result of electrostatic repulsions between His19 and pre-protonated His27. Crucially, we provide an atomic-level understanding of the symmetric proton conduction by identifying pre-activating channel hydration in the C-terminal portion. This research advances our understanding of the function of BM2 function and lays the groundwork for further chemically reactive modeling of the explicit proton transport process as well as possible anti-flu drug design efforts.

Systematic computer-aided disulfide design as a general strategy to stabilize prefusion class I fusion proteins.

Numerous enveloped viruses, such as coronaviruses, influenza, and respiratory syncytial virus (RSV), utilize class I fusion proteins for cell entry. During this process, the proteins transition from a prefusion to a postfusion state, undergoing substantial and irreversible conformational changes. The prefusion conformation has repeatedly shown significant potential in vaccine development. However, the instability of this state poses challenges for its practical application in vaccines. While non-native disulfides have been effective in maintaining the prefusion structure, identifying stabilizing disulfide bonds remains an intricate task. Here, we present a general computational approach to systematically identify prefusion-stabilizing disulfides. Our method assesses the geometric constraints of disulfide bonds and introduces a ranking system to estimate their potential in stabilizing the prefusion conformation. We hypothesized that disulfides restricting the initial stages of the conformational switch could offer higher stability to the prefusion state than those preventing unfolding at a later stage. The implementation of our algorithm on the RSV F protein led to the discovery of prefusion-stabilizing disulfides that supported our hypothesis. Furthermore, the evaluation of our top design as a vaccine candidate in a cotton rat model demonstrated robust protection against RSV infection, highlighting the potential of our approach for vaccine development.

Somatic RAP1B gain-of-function variant underlies isolated thrombocytopenia and immunodeficiency.

The ubiquitously expressed small GTPase Ras-related protein 1B (RAP1B) acts as a molecular switch that regulates cell signaling, cytoskeletal remodeling, and cell trafficking and activates integrins in platelets and lymphocytes. The residue G12 in the P-loop is required for the RAP1B-GTPase conformational switch. Heterozygous germline RAP1B variants have been described in patients with syndromic thrombocytopenia. However, the causality and pathophysiological impact remained unexplored. We report a boy with neonatal thrombocytopenia, combined immunodeficiency, neutropenia, and monocytopenia caused by a heterozygous de novo single nucleotide substitution, c.35G>A (p.G12E) in RAP1B. We demonstrate that G12E and the previously described G12V and G60R were gain-of-function variants that increased RAP1B activation, talin recruitment, and integrin activation, thereby modifying late responses such as platelet activation, T cell proliferation, and migration. We show that in our patient, G12E was a somatic variant whose allele frequency decreased over time in the peripheral immune compartment, but remained stable in bone marrow cells, suggesting a differential effect in distinct cell populations. Allogeneic hematopoietic stem cell transplantation fully restored the patient's hemato-immunological phenotype. Our findings define monoallelic RAP1B gain-of-function variants as a cause for constitutive immunodeficiency and thrombocytopenia. The phenotypic spectrum ranged from isolated hematological manifestations in our patient with somatic mosaicism to complex syndromic features in patients with reported germline RAP1B variants.

Cooperative binding of bivalent ligands yields new insights into the guanidine-II riboswitch.

Riboswitches are involved in regulating the gene expression in bacteria. They are located within the untranslated regions of bacterial messenger RNA and function as switches by adjusting their shape, depending on the presence or absence of specific ligands. To decipher the fundamental aspects of bacterial gene control, it is therefore important to understand the mechanisms that underlie these conformational switches. To this end, a combination of an experimental binding study, molecular simulations and machine learning has been employed to obtain insights into the conformational changes and structural dynamics of the guanidine-II riboswitch. By exploiting the design of a bivalent ligand, we were able to study ligand binding in the aptamer dimer at the molecular level. Spontaneous ligand-binding events, which are usually difficult to simulate, were observed and the contributing factors are described. These findings were further confirmed by in vivo experiments, where the cooperative binding effects of the bivalent ligands resulted in increased binding affinity compared to the native guanidinium ligand. Beyond ligand binding itself, the simulations revealed a novel, ligand-dependent base-stacking interaction outside of the binding pocket that stabilizes the riboswitch.

Dual-Mode Arginine Assay Based on the Conformation Switch of a Ferrocene-Grafted Polypeptide.

Both controllable regulation of the conformational structure of a polypeptide and specific recognition of an amino acid are still arduous challenges. Here, a novel dual-mode (electrochemical and colorimetric) biosensor was built for arginine (Arg) recognition based on a conformation switch, utilizing controllable and synergistic self-assembly of a ferrocene-grafted hexadecapeptide (P16Fc) with gold nanoparticles (AuNPs). Benefiting from the flexibility and unique topological structure of P16Fc formed nanospheres, the assembly and disassembly can undergo a conformation transition induced by Arg through controlling the distance and number of Fc detached from the gold surface, producing on-off electrical signals. Also, they can induce aggregation and dispersion of AuNPs in solution, causing a color change. The mechanism of Arg recognition with polypeptide conformation regulation was well explored by combining microstructure characterizations with molecular mechanics calculations. The electrochemical and colorimetric assays for Arg were successfully established in sensitive and selective manner, not only obtaining a very low detection limit, but also effectively eliminating the interference from other amino acids and overcoming the limitation of AuNP aggregation. Notably, the conformational change-based assay with the peptide regulated by the target will make a powerful tool for the amino acid biosensing and health diagnosis.

Conformational Switch of a Peptide Provides a Novel Strategy to Design Peptide Loaded Porous Organic Polymer for Pyroptosis Pathway Mediated Cancer Therapy.

While peptide-based drug development is extensively explored, this strategy has limitations due to rapid excretion from the body (or shorter half-life in the body) and vulnerability to protease-mediated degradation. To overcome these limitations, a novel strategy for the development of a peptide-based anticancer agent is introduced, utilizing the conformation switch property of a chameleon sequence stretch (PEP1) derived from a mycobacterium secretory protein, MPT63. The selected peptide is then loaded into a new porous organic polymer (PG-DFC-POP) synthesized using phloroglucinol and a cresol derivative via a condensation reaction to deliver the peptide selectively to cancer cells. Utilizing ensemble and single-molecule approaches, this peptide undergoes a transition from a disordered to an alpha-helical conformation, triggered by the acidic environment within cancer cells that is demonstrated. This adopted alpha-helical conformation resulted in the formation of proteolysis-resistant oligomers, which showed efficient membrane pore-forming activity selectively for negatively charged phospholipids accumulated in cancer cell membranes. The experimental results demonstrated that the peptide-loaded PG-DFC-POP-PEP1 exhibited significant cytotoxicity in cancer cells, leading to cell death through the Pyroptosis pathway, which is established by monitoring numerous associated events starting from lysosome membrane damage to GSDMD-induced cell membrane demolition. This novel conformational switch-based drug design strategy is believed to have great potential in endogenous environment-responsive cancer therapy and the development of future drug candidates to mitigate cancers.

De novo DNA-based catch bonds.

All primary chemical interactions weaken under mechanical stress, which imposes fundamental mechanical limits on the materials constructed from them. Biological materials combine plasticity with strength, for which nature has evolved a unique solution-catch bonds, supramolecular interactions that strengthen under tension. Biological catch bonds use force-gated conformational switches to convert weak bonds into strong ones. So far, catch bonds remain exclusive to nature, leaving their potential as mechanoadaptive elements in synthetic systems untapped. Here we report the design and realization of artificial catch bonds. Starting from a minimal set of thermodynamic design requirements, we created a molecular motif capable of catch bonding. It consists of a DNA duplex featuring a cryptic domain that unfolds under tension to strengthen the interaction. We show that these catch bonds recreate force-enhanced rolling adhesion, a hallmark feature of biological catch bonds in bacteria and leukocytes. This Article introduces catch bonds into the synthetic domain, and could lead to the creation of artificial catch-bonded materials.

Effects of an external static EF on the conformational transition of 5-HT1A receptor: A molecular dynamics simulation study

The serotonin receptor subtype 1A (5-HT1AR), one of the G-protein-coupled receptor (GPCR) family, has been implicated in several neurological conditions. Understanding the activation and inactivation mechanism of 5-HT1AR at the molecular level is critical for discovering novel therapeutics in many diseases. Recently there has been a growing appreciation for the role of external electric fields (EFs) in influencing the structure and activity of biomolecules. In this study, we used molecular dynamics (MD) simulations to examine conformational features of active states of 5-HT1AR and investigate the effect of an external static EF with 0.02 V/nm applied on the active state of 5-HT1AR. Our results showed that the active state of 5-HT1AR maintained the native structure, while the EF led to structural modifications in 5-HT1AR, particularly inducing the inward movement of transmembrane helix 6 (TM6). Furthermore, it disturbed the conformational switches associated with activation in the CWxP, DRY, PIF, and NPxxY motifs, consequently predisposing an inclination towards the inactive-like conformation. We also found that the EF led to an overall increase in the dipole moment of 5-HT1AR, encompassing TM6 and pivotal amino acids. The analyses of conformational properties of TM6 showed that the changed secondary structure and decreased solvent exposure occurred upon the EF condition. The interaction of 5-HT1AR with the membrane lipid bilayer was also altered under the EF. Our findings reveal the molecular mechanism underlying the transition of 5-HT1AR conformation induced by external EFs, which offer potential novel insights into the prospect of employing structure-based EF applications for GPCRs.

Molecular mechanism of the ischemia-induced regulatory switch in mammalian complex I.

Respiratory complex I is an efficient driver for oxidative phosphorylation in mammalian mitochondria, but its uncontrolled catalysis under challenging conditions leads to oxidative stress and cellular damage. Ischemic conditions switch complex I from rapid, reversible catalysis into a dormant state that protects upon reoxygenation, but the molecular basis for the switch is unknown. We combined precise biochemical definition of complex I catalysis with high-resolution cryo-electron microscopy structures in the phospholipid bilayer of coupled vesicles to reveal the mechanism of the transition into the dormant state, modulated by membrane interactions. By implementing a versatile membrane system to unite structure and function, attributing catalytic and regulatory properties to specific structural states, we define how a conformational switch in complex I controls its physiological roles.

An α-ketoglutarate conformational switch controls iron accessibility, activation, and substrate selection of the human FTO protein

The fat mass and obesity-associated fatso (FTO) protein is a member of the Alkb family of dioxygenases and catalyzes oxidative demethylation of N6-methyladenosine (m6A), N1-methyladenosine (m1A), 3-methylthymine (m3T), and 3-methyluracil (m3U) in single-stranded nucleic acids. It is well established that the catalytic activity of FTO proceeds via two coupled reactions. The first reaction involves decarboxylation of alpha-ketoglutarate (αKG) and formation of an oxyferryl species. In the second reaction, the oxyferryl intermediate oxidizes the methylated nucleic acid to reestablish Fe(II) and the canonical base. However, it remains unclear how binding of the nucleic acid activates the αKG decarboxylation reaction and why FTO demethylates different methyl modifications at different rates. Here, we investigate the interaction of FTO with 5-mer DNA oligos incorporating the m6A, m1A, or m3T modifications using solution NMR, molecular dynamics (MD) simulations, and enzymatic assays. We show that binding of the nucleic acid to FTO activates a two-state conformational equilibrium in the αKG cosubstrate that modulates the O2 accessibility of the Fe(II) catalyst. Notably, the substrates that provide better stabilization to the αKG conformation in which Fe(II) is exposed to O2 are demethylated more efficiently by FTO. These results indicate that i) binding of the methylated nucleic acid is required to expose the catalytic metal to O2 and activate the αKG decarboxylation reaction, and ii) the measured turnover of the demethylation reaction (which is an ensemble average over the entire sample) depends on the ability of the methylated base to favor the Fe(II) state accessible to O2.

A de novo designed coiled coil-based switch regulates the microtubule motor kinesin-1

Many enzymes are allosterically regulated via conformational change; however, our ability to manipulate these structural changes and control function is limited. Here we install a conformational switch for allosteric activation into the kinesin-1 microtubule motor in vitro and in cells. Kinesin-1 is a heterotetramer that accesses open active and closed autoinhibited states. The equilibrium between these states centers on a flexible elbow within a complex coiled-coil architecture. We target the elbow to engineer a closed state that can be opened with a de novo designed peptide. The alternative states are modeled computationally and confirmed by biophysical measurements and electron microscopy. In cells, peptide-driven activation increases kinesin transport, demonstrating a primary role for conformational switching in regulating motor activity. The designs are enabled by our understanding of ubiquitous coiled-coil structures, opening possibilities for controlling other protein activities.

Plumbagin accelerates serum albumin's amyloid aggregation kinetics and generates fibril polymorphism by inducing non-native β-sheet structures

The ligand-induced conformational switch of proteins has great significance in understanding the biophysics and biochemistry of their self-assembly. In this work, we have investigated the ability of plumbagin (PL), a hydroxynaphthoquinone compound found in the root of the medicinal plant Plumbago zeylanica, to modulate aggregation precursor state, aggregation kinetics and generate distinct fibril of human serum albumin (HSA). PL was found to moderately bind (binding constant Ka ∼ 10−4 M−1)) to domain-II of HSA in the stoichiometric ratio of 1:1. We found that PL-HSA complex aggregation was accelerated as compared to that of HSA aggregation and it may be through an independent pathway. We also detected that fibril produced in the presence of PL is wider in diameter, contains a higher amount of β-sheet (∼18%) and disordered (∼46%) structures, and is less stable. We concluded that the acceleration of aggregation reaction and generation of fibril polymorphism was mainly because of the higher extent of unfolding and high content of non-native β-sheet structure in the aggregation precursor state of PL-HSA complex. This study offers opportunities to explore the ability of ligand binding to modulate aggregation reactions and generate polymorphic protein fibrils.

Working title: Molecular involvement of p53-MDM2 interactome in gastrointestinal cancers.

The interaction between murine double minute 2 (MDM2) and p53, marked by transcriptional induction and feedback inhibition, orchestrates a functional loop dictating cellular fate. The functional loop comprising p53-MDM2 axis is made up of an interactome consisting of approximately 81 proteins, which are spatio-temporally regulated and involved in DNA repair mechanisms. Biochemical and genetic alterations of the interactome result in dysregulation of the p53-mdm2 axis that leads to gastrointestinal (GI) cancers. A large subset of interactome is well known and it consists of proteins that either stabilize p53 or MDM2 and proteins that target the p53-MDM2 complex for ubiquitin-mediated destruction. Upstream signaling events brought about by growth factors and chemical messengers invoke a wide variety of posttranslational modifications in p53-MDM2 axis. Biochemical changes in the transactivation domain of p53 impact the energy landscape, induce conformational switching, alter interaction potential and could change solubility of p53 to redefine its co-localization, translocation and activity. A diverse set of chemical compounds mimic physiological effectors and simulate biochemical modifications of the p53-MDM2 interactome. p53-MDM2 interactome plays a crucial role in DNA damage and repair process. Genetic aberrations in the interactome, have resulted in cancers of GI tract (pancreas, liver, colorectal, gastric, biliary, and esophageal). We present in this article a review of the overall changes in the p53-MDM2 interactors and the effectors that form an epicenter for the development of next-generation molecules for understanding and targeting GI cancers.

Characterization of the small Arabidopsis thaliana GTPase and ADP-ribosylation factor-like 2 protein TITAN 5.

Small GTPases function by conformational switching ability between GDP- and GTP-bound states in rapid cell signaling events. The ADP-ribosylation factor (ARF) family is involved in vesicle trafficking. Though evolutionarily well conserved, little is known about ARF and ARF-like GTPases in plants. Here, we characterized biochemical properties and cellular localization of the essential small ARF-like GTPase TITAN 5/HALLIMASCH/ARL2/ARLC1 (hereafter termed TTN5) from Arabidopsis thaliana. Two TTN5 variants were included in the study with point mutations at conserved residues, suspected to be functional for nucleotide exchange and GTP hydrolysis, TTN5T30N and TTN5Q70L. We found that TTN5 had a very rapid intrinsic nucleotide exchange capacity with a conserved nucleotide switching mechanism. TTN5 acted as a non-classical small GTPase with a remarkably low GTP hydrolysis activity, suggesting it is likely present in GTP-loaded active form in the cell. We analyzed signals from yellow fluorescent protein (YFP)-tagged TTN5 and from in situ immunolocalization of hemagglutine-tagged HA3-TTN5 in Arabidopsis seedlings and in a transient expression system. Together with colocalization using endomembrane markers and pharmacological treatments the microscopic analysis suggests that TTN5 can be present at the plasma membrane and dynamically associated with membranes of vesicles, Golgi stacks and multivesicular bodies. While the TTN5Q70L variant showed similar GTPase activities and localization behavior as wild-type TTN5, the TTN5T30N mutant differed in some aspects. Hence, the unusual capacity of rapid nucleotide exchange activity of TTN5 is linked with cell membrane dynamics, likely associated with vesicle transport pathways in the endomembrane system.

Band Alignment Modulated Polarity-Switchable PEC Ratiometric Sensor through Coupling a pH-Responsive CuTCPP MOF with i-Motif Sensing tool.

In conventional ratiometric photoelectrochemical (PEC) sensors, the detection and reference signals are output sequentially from two independent photosensitive materials. In such a "two-to-two" ratiometric mode, unavoidable difference during dual-interface modification exists, resulting in questionable ratiometric signals and detection results. To address this issue, we propose a novel "one-to-two" ratiometric PEC sensor on a single electrode interface through pH-modulated band alignment engineering. The double ratiometric signals are generated by the synergistic action of a pH-responsive CuTCPP/WS2 photoelectric substrate material and the i-motif sensing tool. Specifically, a ternary heterostructure to generate a photoanodic detection signal is formed under alkaline conditions between CuTCPP/WS2 and signal label CdS QDs binding to the i-motif. While under acidic conditions, a photocurrent polarity conversion and signaling labels detachment, induced by the band realignment of CuTCPP/WS2 and the i-motif conformational switching, produce a reliable internal reference photocathodic signal. The feasibility of this two-wing signal generation strategy is validated by detecting mycotoxin ochratoxin A, which achieves accurate and reliable ratio detection results. Overall, this work provides guidance for the design of a PEC ratiometric determination system and exhibits great potential to be applied in practical analysis research.