Construction of CDs@ß-CD@CCM ratiometric fluorescence probe for FRET-based ClO--sensing.

ß-Cyclodextrin (ß-CD) -functionalized carbon quantum dots (CDs) loaded with curcumin (CCM) were used for ClO--sensing with high sensitivity and selectivity. This fluorescence resonance energy transfer (FRET)-based sensor was created through attaching CCM to the CDs via ß-CD linker. CCM could get into the interior of ß-CD triggering the FRET from CDs to CCM, providing an "off" state of the CDs. However, the effect of FRET was weakened by the ClO-, because the o- methoxyphenol structure from CCM was oxidized to be benzoquinone. The fluorescence intensity of CDs@ß-CD@CCM at 440 nm can be heightened and 520 nm from CCM can decrease along with the increased ClO-. Therefore, a ratiometric fluorescence probe for ClO--sensing is successfully constructed. It conforms to a polynomial curve equation which is I440/I520 = -0.0268+0.0315 CClO-+0.0055[CClO-]2 (R2= 0.9958) between 0-18.4 µM ClO-. Furthermore, we also obtain excellent results using this spectrophotometric method for ClO-- sensing in pure water and commercial disinfectants, which afford potential in the environment monitoring area. We expect this sensing platform could be helpful in other analogous probes in relevant fields.

Subtype-specific conformational landscape of NMDA receptor gating.

N-methyl-D-aspartate receptors are ionotropic glutamate receptors that mediate synaptic transmission and plasticity. Variable GluN2 subunits in diheterotetrameric receptors with identical GluN1 subunits set very different functional properties. To understand this diversity, we use single-molecule fluorescence resonance energy transfer (smFRET) to measure the conformations of the ligand binding domain and modulatory amino-terminal domain of the common GluN1 subunit in receptors with different GluN2 subunits. Our results demonstrate a strong influence of the GluN2 subunits on GluN1 rearrangements, both in non-agonized and partially agonized activation intermediates, which have been elusive to structural analysis, and in the fully liganded state. Chimeric analysis reveals structural determinants that contribute to these subtype differences. Our study provides a framework for understanding the conformational landscape that supports highly divergent levels of activity, desensitization, and agonist potency in receptors with different GluN2s and could open avenues for the development of subtype-specific modulators.

Excitation/emission-enhanced heterostructure photonic crystal array synergizing with "DD-A" FRET entropy-driven circuit for high-resolution and ultrasensitive analysis of ctDNA.

Circulating tumor DNA (ctDNA) is an emerging biomarker of liquid biopsy for cancer. But it remains a challenge to achieve simple, sensitive and specific detection of ctDNA because of low abundance and single-base mutation. In this work, an excitation/emission-enhanced heterostructure photonic crystal (PC) array synergizing with entropy-driven circuit (EDC) was developed for high-resolution and ultrasensitive analysis of ctDNA. The donor donor-acceptor FÖrster resonance energy transfer ("DD-A" FRET) was integrated in EDC based on the introduction of simple auxiliary strand, which exhibited higher sensitivity than that of traditional EDC. The heterostructure PC array was constructed with the bilayer periodic nanostructures of nanospheres. Because the heterostructure PC has the adjustable dual photonic band gaps (PBGs) by changing nanosphere sizes, and the "DD-A" FRET can offer the excitation and emission peak with enough distance, it helps the successful matches between the dual PBGs of heterostructure PC and the excitation/emission peaks of "DD-A" FRET; thus, the fluorescence from EDC can be enhanced effectively from both of excitation and emission processes on heterostructure PC array. Besides, high-resolution of single-base mutation was obtained through the strict recognition of EDC. Benefiting from the specific spectrum-matched and synergetic amplification of heterostructure PC and EDC with "DD-A" FRET, the proposed array obtained ultrasensitive detection of ctDNA with LOD of 12.9 fM, and achieved the analysis of mutation frequency as low as 0.01%. Therefore, the proposed strategy has the advantages of simple operation, mild conditions (enzyme-free and isothermal), high-sensitivity, high-resolution and high-throughput analysis, showing potential in bioassay and clinical application.

Development of a Time-Resolved Fluorescence Resonance Energy Transfer ultra-high throughput screening assay targeting SYK and FCER1G interaction.

The spleen tyrosine kinase (SYK) and high affinity immunoglobulin epsilon receptor subunit gamma (FCER1G) interaction has a major role in the normal innate and adaptive immune responses, but dysregulation of this interaction is implicated in several human diseases, including autoimmune disorders, hematological malignancies, and Alzheimer's Disease. Development of small molecule chemical probes could aid in studying this pathway both in normal and aberrant contexts. Herein, we describe the miniaturization of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay to measure the interaction between SYK and FCER1G in a 1536-well ultrahigh throughput screening (uHTS) format. The assay utilizes the His-SH2 domains of SYK, which are indirectly labeled with anti-His-terbium to serve as a TR-FRET donor and a FITC-conjugated phosphorylated ITAM domain peptide of FCER1G to serve as an acceptor. We have optimized the assay into a 384-well HTS format and further miniaturized the assay into a 1536-well uHTS format. Robust assay performance has been achieved with a Z' factor > 0.8 and signal-to-background (S/B) ratio > 15. The utilization of this uHTS TR-FRET assay for compound screening has been validated by a pilot screening of 2,036 FDA-approved and bioactive compounds library. Several primary hits have been identified from the pilot uHTS. One compound, hematoxylin, was confirmed to disrupt the SYK/FECR1G interaction in an orthogonal protein-protein interaction assay. Thus, our optimized and miniaturized uHTS assay could be applied to future scaling up of a screening campaign to identify small molecule inhibitors targeting the SYK and FCER1G interaction.

Demystifying Trion Emission in CdSe Nanoplatelets.

At cryogenic temperatures, the photoluminescence spectrum of CdSe nanoplatelets (NPLs) usually consists of multiple emission lines, the origin of which is still under debate. While there seems to be consensus that both neutral excitons and trions contribute to the NPL emission, the prominent role of trions is rather puzzling. In this work, we demonstrate that Förster resonant energy transfer in stacks of NPLs combined with hole trap states in specific NPLs within the stack trigger trion formation, while single NPL spectra are dominated by neutral excitonic emission. This interpretation is verified by implementing copper (Cu+) dopants into the lattice as intentional hole traps. Trion emission gets strongly enhanced, and due to the large amount of hole trapping Cu+ states in each single NPL, trion formation does not necessarily require stacking of NPLs. Thus, the ratio between trion and neutral exciton emission can be controlled by either changing the amount of stacked NPLs during sample preparation or implementing copper dopants into the lattice which act as additional hole traps.

Pilot-Scale Screening of Clinically Approved Drugs to Identify Uridine Insertion/Deletion RNA Editing Inhibitors in Trypanosoma brucei.

RNA editing pathway is a validated target in kinetoplastid parasites (Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp.) that cause severe diseases in humans and livestock. An essential large protein complex, the editosome, mediates uridine insertion and deletion in RNA editing through a stepwise process. This study details the discovery of editosome inhibitors by screening a library of widely used human drugs using our previously developed in vitro biochemical Ribozyme Insertion Deletion Editing (RIDE) assay. Subsequent studies on the mode of action of the identified hits and hit expansion efforts unveiled compounds that interfere with RNA-editosome interactions and novel ligase inhibitors with IC50 values in the low micromolar range. Docking studies on the ligase demonstrated similar binding characteristics for ATP and our novel epigallocatechin gallate inhibitor. The inhibitors demonstrated potent trypanocidal activity and are promising candidates for drug repurposing due to their lack of cytotoxic effects. Further studies are necessary to validate these targets using more definitive gene-editing techniques and to enhance the safety profile.

Luminescence "Turn-On" Sensing of Brain Natriuretic Peptide (BNP) - Dilated Cardiomyopathy Biomarker Based on the MoS2 Nanosheet Quenched Terbium Citrate Complex.

Dilated cardiomyopathy (DCM), known as myocardial metabolic dysfunction, is recognized as a clinical condition characterized by left ventricular dilation or improper contraction of cardiac muscles in the absence of coronary atherosclerosis and hypertension. It is an independent risk factor for cardiac function caused by a hyperglycemic condition in diabetic patients leading to heart failure (HF), which renders the early diagnosis of DCM highly challenging. Hence, detection of early diagnostic biomarkers in blood serum to identify DCM conditions is quite requisite. Brain natriuretic peptide (BNP) is a well-recognized biomarker for heart failure and reported as an early diagnostic biomarker for DCM. In this work, we developed a terbium citrate based MoS2 nanosheet (NS) coupled immunoprobe for the sensitive detection of BNP. The antibody conjugated Tb-citrate complex exhibits green fluorescence, which is quenched by the introduction of MoS2 NS. On subsequent addition of antigen BNP, the fluorescence is enhanced because of specific antigen-antibody interaction. The probe is selective and sensitive toward BNP in a linear range from 30.76 to 849.85 pg/mL with a low LOD of 3.87 pg/mL. The probe is validated in spiked human serum samples with good recovery percentage.

Methods for Studying Fusion of Bacterial Extracellular Vesicles with Intact Bacteria and Host Cells.

Bacterial extracellular vesicles (BEVs) are nano- or micrometer-sized membrane-bound lipid vesicles released from both Gram-negative and Gram-positive bacteria. Cellular transport, communication, pathogenesis, and host-pathogen interactions are some of the major biological processes impacted by BEVs. Among these, host-pathogen interactions and bacterial pathogenesis are emerging as highly important targetable avenues underlined by the issues of antimicrobial resistance, thus demanding novel targets and approaches to treat bacterial infections. In this aspect, the study of the interaction of BEVs with bacteria and/or host cells becomes imperative and brings the membrane fusion process to the forefront. Furthermore, membrane fusion also underscores the performance of BEVs as nano-therapeutic delivery platforms. Here, we report methods to study fusion kinetics between mycobacteria-derived extracellular vesicles, which we refer to as MEVs, and intact mycobacteria or MEVs themselves. We also discuss the isolation of MEVs and their characterization. We outline critical factors that affect fusion kinetics by MEVs. The same principle can be extended for studying fusion between BEVs and mammalian host cells important for understanding how BEVs influence host-pathogen crosstalk.

DNAJA2 and Hero11 mediate similar conformational extension and aggregation suppression of TDP-43.

Many RNA binding proteins (RBPs) contain low-complexity domains (LCDs) with prion-like compositions. These long intrinsically disordered regions regulate their solubility, contributing to their physiological roles in RNA processing and organization. However, this also makes these RBPs prone to pathological misfolding and aggregation that are characteristic of neurodegenerative diseases. For example, TAR DNA-binding protein 43 (TDP-43) forms pathological aggregates associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). While molecular chaperones are well-known suppressors of these aberrant events, we recently reported that highly disordered, hydrophilic and charged heat-resistant obscure (Hero) proteins may have similar effects. Specifically, Hero proteins can maintain the activity of other proteins from denaturing conditions in vitro, while their overexpression can suppress cellular aggregation and toxicity associated with aggregation-prone proteins. However, it is unclear how these protective effects are achieved. Here, we utilized single-molecule FRET to monitor the conformations of the aggregation-prone prion-like LCD of TDP-43. While we observed high conformational heterogeneity in wild-type LCD, the ALS-associated mutation A315T promoted collapsed conformations. In contrast, an Hsp40 chaperone, DNAJA2, and a Hero protein, Hero11 stabilized extended states of the LCD, consistent with their ability to suppress the aggregation of TDP-43. Our results link single-molecule effects on conformation to macro effects on bulk aggregation, where a Hero protein, like a chaperone, can maintain the conformational integrity of a client protein to prevent its aggregation.

A cancer-targeted glutathione-gated probe for self-sufficient ST/CDT combination therapy and FRET-based miRNA imaging.

A cancer-targeted glutathione (GSH)-gated theranostic probe (CGT probe) for intracellular miRNA imaging and combined treatment of self-sufficient starvation therapy (ST) and chemodynamic therapy (CDT) was developed. The CGT probe is constructed using MnO2 nanosheet (MS) as carrier material to adsorb the elaborately designed functional DNAs. It can be internalized by cancer cells via specific recognition between the AS1411 aptamer and nucleolin. After CGT probe entering the cancer cells, the overexpressed GSH, as gate-control, can degrade MS to Mn2+ which can be used for CDT by Fenton-like reaction. Simultaneously, Mn2+-mediated CDT can further cascade with the enzyme-like activities (catalase-like activity and glucose oxidase-like activity) of CGT probe, achieving self-sufficient ST/CDT synergistic therapy. Meanwhile, the anchored DNAs are released, achieving in situ signal amplification via disubstituted-catalytic hairpin assembly (DCHA) and FRET (fluorescence resonance energy transfer) imaging of miR-21. The in vitro and in vivo experiments demonstrated that accurate and sensitive miRNA detection can be achieved using the CGT probe. Overall, the ingenious CGT probe opens a new avenue for the development of early clinical diagnosis and cancer therapy.

Efficient enrichment of free target sequences in an integrated microfluidic device for point-of-care detection systems.

Nucleic acid biomarker detection has great importance in the diagnosis of disease, the monitoring of disease progression and the classification of patients according to treatment decision making. Nucleic acid biomarkers found in the blood of patients have generated a lot of interest due to the possibility of being detected non-invasively which makes them ideal for monitoring and screening tests and particularly amenable to point-of-care (POC) or self-testing. A major challenge to POC molecular diagnostics is the need to enrich the target to optimise detection. In this work, we describe a microfabricated device for the enrichment of short dsDNA target sequences, which is especially valuable for potential detection methods, as it improves the probability of effectively detecting the target in downstream analyses. The device integrated a heating element and a temperature sensor with a microfluidic chamber to carry out the denaturation of the dsDNA combined with blocking-probes to enrich the target. This procedure was validated by fluorescence resonance energy transfer (FRET) technique, labelling DNA with a fluorophore and a quencher. As proof of concept, a 23-mer long dsDNA sequence corresponding to the L858R mutation of the EGFR gene was used. The qualitative results obtained determined that the most optimal blocking rate was obtained with the incorporation of 11/12-mer blocking-probes at a total concentration of 6 µM. This device is a powerful DNA preparation tool, which is an indispensable initial step for subsequent detection of sequences via nucleic acid hybridisation methods.

Using lipid binding proteins and advanced microscopy to study lipid domains.

Sphingomyelin is postulated to form clusters with glycosphingolipids, cholesterol and other sphingomyelin molecules in biomembranes through hydrophobic interaction and hydrogen bonds. These clusters form submicron size lipid domains. Proteins that selectively binds sphingomyelin and/or cholesterol are useful to visualize the lipid domains. Due to their small size, visualization of lipid domains requires advanced microscopy techniques in addition to lipid binding proteins. This Chapter describes the method to characterize plasma membrane sphingomyelin-rich and cholesterol-rich lipid domains by quantitative microscopy. This Chapter also compares different permeabilization methods to visualize intracellular lipid domains.

Highly Sensitive and Selective Fluorescence and Smartphone-Based Sensor for Detection of Rutin Using Boron Nitrogen Co-doped Graphene Quantum Dots.

This research explores the fluorescence properties and photostability of boron nitrogen co-doped graphene quantum dots (BN-GQDs), evaluating their effectiveness as sensors for rutin (RU). BN-GQDs are biocompatible and exhibit notable absorbance and fluorescence characteristics, making them suitable for sensing applications. The study utilized various analytical techniques to investigate the chemical composition, structure, morphology, optical attributes, elemental composition, and particle size of BN-GQDs. Techniques included X-ray diffraction (XRD), energy-dispersive X-ray spectroscopy (EDS), transmission electron microscopy (TEM), scanning electron microscopy (SEM), and atomic force microscopy (AFM). The average particle size of the BN-GQDs was determined to be approximately 3.5 ± 0.3 nm. A clear correlation between the emission intensity ratio and RU concentration was identified across the range of 0.42 to 4.1 µM, featuring an impressively low detection limit (LOD) of 1.23 nM. The application of BN-GQDs as fluorescent probes has facilitated the development of a highly sensitive and selective RU detection method based on Förster resonance energy transfer (FRET) principles. This technique leverages emission at 465 nm. Density Functional Theory (DFT) analyses confirm that FRET is the primary mechanism behind fluorescence quenching, as indicated by the energy levels of the lowest unoccupied molecular orbitals (LUMOs) of BN-GQDs and RU. The method's effectiveness has been validated by measuring RU concentrations in human serum samples, showing a recovery range between 97.8% and 103.31%. Additionally, a smartphone-based detection method utilizing BN-GQDs has been successfully implemented, achieving a detection limit (LOD) of 49 nM.

Fluorescence Studies on the Binding Affinity and Determination of Vitamin B12 in the Presence of Fibrinogen.

The binding properties between vitamin B12 (vitB12, cyanocobalamin) and fibrinogen (Fib) were investigated by UV-vis absorption and steady-state/three-dimentional (3D) fluorescence spectra techniques as well as molecular docking. The experimental results showed that the intrinsic fluorescence of Fib quenched by vitB12 with static mechanism to form a non-fluorescent complex. The positive signs of thermodynamic parameters, ΔH (92.18 kJ/mol) and ΔS (433.5 J/molK), indicated that the hydrophobic forces were dominant in the binding mode. The molecular docking data were found to be in agreement with these experimental results and were confirmed by three hydrophobic interactions between the Trp430, Try390 residues of Fib and the vitamin. 3D spectra showed that fibrinogen undergoes a conformation change when it interacts with vitB12. Based on non-radiative energy transfer theory, binding distance was calculated to be 3.94 nm between donor (tryptophan residues of Fib) and acceptor (vitB12). The limit of detection (LOD) of vitB12 was calculated as 2.08 µM in the presence of fibrinogen. The relative standard deviation (RSD) of method was 4.28% for determinations (n = 7) of a vitB12 solution with the concentration of 7.80 µM.

GA dynamics governing nodulation revealed using GIBBERELLIN PERCEPTION SENSOR 2 in Medicago truncatula lateral organs.

During nutrient scarcity, plants can adapt their developmental strategy to maximize their chance of survival. Such plasticity in development is underpinned by hormonal regulation, which mediates the relationship between environmental cues and developmental outputs. In legumes, endosymbiosis with nitrogen fixing bacteria (rhizobia) is a key adaptation for supplying the plant with nitrogen in the form of ammonium. Rhizobia are housed in lateral root-derived organs termed nodules that maintain an environment conducive to Nitrogenase in these bacteria. Several phytohormones are important for regulating the formation of nodules, with both positive and negative roles proposed for gibberellin (GA). In this study, we determine the cellular location and function of bioactive GA during nodule organogenesis using a genetically-encoded second generation GA biosensor, GIBBERELLIN PERCEPTION SENSOR 2 in Medicago truncatula. We find endogenous bioactive GA accumulates locally at the site of nodule primordia, increasing dramatically in the cortical cell layers, persisting through cell divisions and maintaining accumulation in the mature nodule meristem. We show, through mis-expression of GA catabolic enzymes that suppress GA accumulation, that GA acts as a positive regulator of nodule growth and development. Furthermore, increasing or decreasing GA through perturbation of biosynthesis gene expression can increase or decrease the size of nodules, respectively. This is unique from lateral root formation, a developmental program that shares common organogenesis regulators. We link GA to a wider gene regulatory program by showing that nodule-identity genes induce and sustain GA accumulation necessary for proper nodule formation.

Application of FRET- and BRET-based live-cell biosensors in deorphanization and ligand discovery studies on orphan G protein-coupled receptors.

Bioluminescence- and fluorescence-based resonance energy transfer assays have gained considerable attention in pharmacological research as high-throughput scalable tools applicable to drug discovery. To this end, G protein-coupled receptors represent the biggest target class for marketed drugs, and among them, orphan G protein-coupled receptors have the biggest untapped therapeutic potential. In this review, the cases where biophysical methods, BRET and FRET, were employed for deorphanization and ligand discovery studies on orphan G protein-coupled receptors are listed and discussed.

A wearable microneedle patch incorporating reversible FRET-based hydrogel sensors for continuous glucose monitoring.

Continuous glucose monitors are crucial for diabetes management, but invasive sampling, signal drift and frequent calibrations restrict their widespread usage. Microneedle sensors are emerging as a minimally-invasive platform for real-time monitoring of clinical parameters in interstitial fluid. Herein, a painless and flexible microneedle sensing patch is constructed by a mechanically-strong microneedle base and a thin layer of fluorescent hydrogel sensor for on-site, accurate, and continuous glucose monitoring. The Förster resonance energy transfer (FRET)-based hydrogel sensors are fabricated by facile photopolymerizations of acryloylated FRET pairs and glucose-specific phenylboronic acid. The optimized hydrogel sensor enables quantification of glucose with reversibility, high selectivity, and signal stability against photobleaching. Poly (ethylene glycol diacrylate)-co-polyacrylamide hydrogel is utilized as the microneedle base, facilitating effective skin piercing and biofluid extraction. The integrated microneedle sensor patch displays a sensitivity of 0.029 mM-1 in the (patho)physiological range, a low detection limit of 0.193 mM, and a response time of 7.7 min in human serum. Hypoglycemia, euglycemia and hyperglycemia are continuously monitored over 6 h simulated meal and rest activities in a porcine skin model. This microneedle sensor with high transdermal analytical performance offers a powerful tool for continuous diabetes monitoring at point-of-care settings.

iMAX FRET (Information Maximized FRET) for Multipoint Single-Molecule Structural Analysis.

Understanding the structure of biomolecules is vital for deciphering their roles in biological systems. Single-molecule techniques have emerged as alternatives to conventional ensemble structure analysis methods for uncovering new biology in molecular dynamics and interaction studies, yet only limited structural information could be obtained experimentally. Here, we address this challenge by introducing iMAX FRET, a one-pot method that allows ab initio 3D profiling of individual molecules using two-color FRET measurements. Through the stochastic exchange of fluorescent weak binders, iMAX FRET simultaneously assesses multiple distances on a biomolecule within a few minutes, which can then be used to reconstruct the coordinates of up to four points in each molecule, allowing structure-based inference. We demonstrate the 3D reconstruction of DNA nanostructures, protein quaternary structures, and conformational changes in proteins. With iMAX FRET, we provide a powerful approach to advance the understanding of biomolecular structure by expanding conventional FRET analysis to three dimensions.

Lattice matching enables construction of CaS@NaYF4 heterostructure with synergistically enhanced water resistance and luminescence for antibiotic detection.

Rare earth (RE)-doped CaS phosphors have been widely used as light-emitting components in various fields. Nevertheless, the application of nanosized CaS particles is still significantly limited by their poor water resistance and weak luminescence. Herein, a lattice-matching strategy is developed by growing an inert shell of cubic NaYF4 phase on the CaS luminescent core. Due to their similarity in crystal structure, a uniform core-shell heterostructure (CaS:Ce3+@NaYF4) can be obtained, which effectively protects the CaS:Ce3+ core from degradation in aqueous environment and enhances its luminescence intensity. As a proof of concept, a label-free aptasensor is further constructed by combining core-shell CaS:Ce3+@NaYF4 and Au nanoparticles (AuNPs) for the ultrasensitive detection of kanamycin antibiotics. Based on the efficient FRET process, the detection linear range of kanamycin spans from 100 to 1000 nM with a detection limit of 7.8 nM. Besides, the aptasensor shows excellent selectivity towards kanamycin antibiotics, and has been successfully applied to the detection of kanamycin spiked in tap water and milk samples, demonstrating its high potential for sensing applications.

Intermediate steps in the formation of neuronal SNARE complexes.

Neuronal exocytosis requires the assembly of three SNARE proteins, syntaxin and SNAP25 on the plasma membrane and synaptobrevin on the vesicle membrane. However, the precise steps in this process and the points at which assembly and fusion are controlled by regulatory proteins are unclear. In the present work, we examine the kinetics and intermediate states during SNARE assembly in vitro using a combination of time resolved fluorescence and EPR spectroscopy. We show that syntaxin rapidly forms a dimer prior to forming the kinetically stable 2:1 syntaxin:SNAP25 complex and that the 2:1 complex is not diminished by the presence of excess SNAP25. Moreover, the 2:1 complex is temperature-dependent with a reduced concentration at 37 °C. The two segments of SNAP25 behave differently. The N-terminal SN1 segment of SNAP25 exhibits a pronounced increase in backbone ordering from the N- to the C-terminus that is not seen in the C-terminal SNAP25 segment SN2. Both the SN1 and SN2 segments of SNAP25 will assemble with syntaxin; however, while the association of the SN1 segment with syntaxin produces a stable 2:2 (SN1:syntaxin) complex, the complex formed between SN2 and syntaxin is largely disordered. Synaptobrevin fails to bind syntaxin alone but will associate with syntaxin in the presence of either the SN1 or SN2 segments; however, the synaptobrevin:syntaxin:SN2 complex remains disordered. Taken together, these data suggest that synaptobrevin and syntaxin do not assemble in the absence of SNAP25 and that the SN2 segment of SNAP25 is the last to enter the SNARE complex.