Stability, folding dynamics, and long-range conformational transition of the synaptic t-SNARE complex.

Synaptic soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) couple their stepwise folding to fusion of synaptic vesicles with plasma membranes. In this process, three SNAREs assemble into a stable four-helix bundle. Arguably, the first and rate-limiting step of SNARE assembly is the formation of an activated binary target (t)-SNARE complex on the target plasma membrane, which then zippers with the vesicle (v)-SNARE on the vesicle to drive membrane fusion. However, the t-SNARE complex readily misfolds, and its structure, stability, and dynamics are elusive. Using single-molecule force spectroscopy, we modeled the synaptic t-SNARE complex as a parallel three-helix bundle with a small frayed C terminus. The helical bundle sequentially folded in an N-terminal domain (NTD) and a C-terminal domain (CTD) separated by a central ionic layer, with total unfolding energy of ∼17 kBT, where kB is the Boltzmann constant and T is 300 K. Peptide binding to the CTD activated the t-SNARE complex to initiate NTD zippering with the v-SNARE, a mechanism likely shared by the mammalian uncoordinated-18-1 protein (Munc18-1). The NTD zippering then dramatically stabilized the CTD, facilitating further SNARE zippering. The subtle bidirectional t-SNARE conformational switch was mediated by the ionic layer. Thus, the t-SNARE complex acted as a switch to enable fast and controlled SNARE zippering required for synaptic vesicle fusion and neurotransmission.

Kinetically coupled folding of a single HIV-1 glycoprotein 41 complex in viral membrane fusion and inhibition.

HIV-1 glycoprotein 41 (gp41) mediates viral entry into host cells by coupling its folding energy to membrane fusion. Gp41 folding is blocked by fusion inhibitors, including the commercial drug T20, to treat HIV/AIDS. However, gp41 folding intermediates, energy, and kinetics are poorly understood. Here, we identified the folding intermediates of a single gp41 trimer-of-hairpins and measured their associated energy and kinetics using high-resolution optical tweezers. We found that folding of gp41 hairpins was energetically independent but kinetically coupled: Each hairpin contributed a folding energy of ∼-23 kBT, but folding of one hairpin successively accelerated the folding rate of the next one by ∼20-fold. Membrane-mimicking micelles slowed down gp41 folding and reduced the stability of the six-helix bundle. However, the stability was restored by cooperative folding of the membrane-proximal external region. Surprisingly, T20 strongly inhibited gp41 folding by actively displacing the C-terminal hairpin strand in a force-dependent manner. The inhibition was abolished by a T20-resistant gp41 mutation. The energetics and kinetics of gp41 folding established by us provides a basis to understand viral membrane fusion, infection, and therapeutic intervention.

Hidden Markov Modeling with Detailed Balance and Its Application to Single Protein Folding.

Hidden Markov modeling (HMM) has revolutionized kinetic studies of macromolecules. However, results from HMM often violate detailed balance when applied to the transitions under thermodynamic equilibrium, and the consequence of such violation has not been well understood. Here, to our knowledge, we developed a new HMM method that satisfies detailed balance (HMM-DB) and optimizes model parameters by gradient search. We used free energy of stable and transition states as independent fitting parameters and considered both normal and skew normal distributions of the measurement noise. We validated our method by analyzing simulated extension trajectories that mimicked experimental data of single protein folding from optical tweezers. We then applied HMM-DB to elucidate kinetics of regulated SNARE zippering containing degenerate states. For both simulated and measured trajectories, we found that HMM-DB significantly reduced overfitting of short trajectories compared to the standard HMM based on an expectation-maximization algorithm, leading to more accurate and reliable model fitting by HMM-DB. We revealed how HMM-DB could be conveniently used to derive a simplified energy landscape of protein folding. Finally, we extended HMM-DB to correct the baseline drift in single-molecule trajectories. Together, we demonstrated an efficient, versatile, and reliable method of HMM for kinetics studies of macromolecules under thermodynamic equilibrium.

Interlinked Microcone Resistive Sensors Based on Self-Assembly Carbon Nanotubes Film for Monitoring of Signals.

Flexible pressure sensors still face difficulties achieving a constantly adaptable micronanostructure of substrate materials. Interlinked microcone resistive sensors were fabricated by polydimethylsiloxane (PDMS) nanocone array. PDMS nanocone array was achieved by the second transferring tapered polymethyl methacrylate (PMMA) structure. In addition, self-assembly 2D carbon nanotubes (CNTs) networks as a conducting layer were prepared by a low-cost, dependable, and ultrafast Langmuir−Blodgett (LB) process. In addition, the self-assembled two-dimensional carbon nanotubes (CNTs) network as a conductive layer can change the internal resistance due to pressure. The results showed that the interlinked sensor with a nanocone structure can detect the external pressure by the change of resistivity and had a sensitive resistance change in the low pressure (<200 Pa), good stability through 2800 cycles, and a detection limit of 10 kPa. Based on these properties, the electric signals were tested, including swallowing throat, finger bending, finger pressing, and paper folding. The simulation model of the sensors with different structural parameters under external pressure was established. With the advantages of high sensitivity, stability, and wide detection range, this sensor shows great potential for monitoring human motion and can be used in wearable devices.

Microdome-Tunable Graphene/Carbon Nanotubes Pressure Sensors Based on Polystyrene Array for Wearable Electronics.

Resistive pressure sensors are appealing due to having several advantages, such as simple reading mechanisms, simple construction, and quick dynamic response. Achieving a constantly changeable microstructure of sensing materials is critical for the flexible pressure sensor and remains a difficulty. Herein, a flexible, tunable resistive pressure sensors is developed via simple, low-cost microsphere self-assembly and graphene/carbon nanotubes (CNTs) solution drop coating. The sensor uses polystyrene (PS) microspheres to construct an interlocked dome microstructure with graphene/CNTs as a conductive filler. The results indicate that the interlocked microdome-type pressure sensor has better sensitivity than the single microdome-type and single planar-type without surface microstructure. The pressure sensor's sensitivity can be adjusted by varying the diameter of PS microspheres. In addition, the resistance of the sensor is also tunable by adjusting the number of graphene/CNT conductive coating layers. The developed flexible pressure sensor effectively detected human finger bending, demonstrating tremendous potential in human motion monitoring.

Single-Molecule Optical Tweezers Study of Regulated SNARE Assembly.

Intracellular membrane fusion mediates material and information exchange among different cells or cellular compartments with high accuracy and spatiotemporal resolution. Fusion is driven by ordered folding and assembly of soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptors (SNAREs) and regulated by many other proteins. Understanding regulated SNARE assembly is key to dissecting mechanisms and physiologies of various fusion processes and their associated diseases. Yet, it remains challenging to study regulated SNARE assembly using traditional ensemble-based experimental approaches. Here, we describe our new method to measure the energy and kinetics of neuronal SNARE assembly in the presence of α-SNAP, using a single-molecule manipulation approach based on high-resolution optical tweezers. Detailed experimental protocols and methods of data analysis are shown. This approach can be widely applied to elucidate the effects of regulatory proteins on SNARE assembly and membrane fusion.

Munc18-1 catalyzes neuronal SNARE assembly by templating SNARE association.

Sec1/Munc18-family (SM) proteins are required for SNARE-mediated membrane fusion, but their mechanism(s) of action remain controversial. Using single-molecule force spectroscopy, we found that the SM protein Munc18-1 catalyzes step-wise zippering of three synaptic SNAREs (syntaxin, VAMP2, and SNAP-25) into a four-helix bundle. Catalysis requires formation of an intermediate template complex in which Munc18-1 juxtaposes the N-terminal regions of the SNARE motifs of syntaxin and VAMP2, while keeping their C-terminal regions separated. SNAP-25 binds the templated SNAREs to induce full SNARE zippering. Munc18-1 mutations modulate the stability of the template complex in a manner consistent with their effects on membrane fusion, indicating that chaperoned SNARE assembly is essential for exocytosis. Two other SM proteins, Munc18-3 and Vps33, similarly chaperone SNARE assembly via a template complex, suggesting that SM protein mechanism is conserved.

Single-Molecule Protein Folding Experiments Using High-Precision Optical Tweezers.

How proteins fold from linear chains of amino acids to delicate three-dimensional structures remains a fundamental biological problem. Single-molecule manipulation based on high-resolution optical tweezers (OT) provides a powerful approach to study protein folding with unprecedented spatiotemporal resolution. In this method, a single protein or protein complex is tethered between two beads confined in optical traps and pulled. Protein unfolding induced by the mechanical force is counteracted by the spontaneous folding of the protein, reaching a dynamic equilibrium at a characteristic force and rate. The transition is monitored by the accompanying extension change of the protein and used to derive conformations and energies of folding intermediates and their associated transition kinetics. Here, we provide general strategies and detailed protocols to study folding of proteins and protein complexes using optical tweezers, including sample preparation, DNA-protein conjugation and methods of data analysis to extract folding energies and rates from the single-molecule measurements.

Single-molecule force spectroscopy of protein-membrane interactions.

Many biological processes rely on protein-membrane interactions in the presence of mechanical forces, yet high resolution methods to quantify such interactions are lacking. Here, we describe a single-molecule force spectroscopy approach to quantify membrane binding of C2 domains in Synaptotagmin-1 (Syt1) and Extended Synaptotagmin-2 (E-Syt2). Syts and E-Syts bind the plasma membrane via multiple C2 domains, bridging the plasma membrane with synaptic vesicles or endoplasmic reticulum to regulate membrane fusion or lipid exchange, respectively. In our approach, single proteins attached to membranes supported on silica beads are pulled by optical tweezers, allowing membrane binding and unbinding transitions to be measured with unprecedented spatiotemporal resolution. C2 domains from either protein resisted unbinding forces of 2-7 pN and had binding energies of 4-14 kBT per C2 domain. Regulation by bilayer composition or Ca2+ recapitulated known properties of both proteins. The method can be widely applied to study protein-membrane interactions.

α-SNAP Enhances SNARE Zippering by Stabilizing the SNARE Four-Helix Bundle.

Intracellular membrane fusion is mediated by dynamic assembly and disassembly of soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptors (SNAREs). α-SNAP guides NSF to disassemble SNARE complexes after membrane fusion. Recent experiments showed that α-SNAP also dramatically enhances SNARE assembly and membrane fusion. How α-SNAP is involved in these opposing activities is not known. Here, we examine the effect of α-SNAP on the stepwise assembly of the synaptic SNARE complex using optical tweezers. We found that α-SNAP destabilized the linker domain (LD) of the SNARE complex but stabilized its C-terminal domain (CTD) through a conformational selection mechanism. In contrast, α-SNAP minimally affected assembly of the SNARE N-terminal domain (NTD), indicating that α-SNAP barely bound the partially assembled trans-SNARE complex. Thus, α-SNAP recognizes the folded CTD for SNARE disassembly with NSF and subtly modulates membrane fusion by altering the stabilities of the SNARE CTD and LD.

Quantifying and optimizing single-molecule switching nanoscopy at high speeds.

Single-molecule switching nanoscopy overcomes the diffraction limit of light by stochastically switching single fluorescent molecules on and off, and then localizing their positions individually. Recent advances in this technique have greatly accelerated the data acquisition speed and improved the temporal resolution of super-resolution imaging. However, it has not been quantified whether this speed increase comes at the cost of compromised image quality. The spatial and temporal resolution depends on many factors, among which laser intensity and camera speed are the two most critical parameters. Here we quantitatively compare the image quality achieved when imaging Alexa Fluor 647-immunolabeled microtubules over an extended range of laser intensities and camera speeds using three criteria - localization precision, density of localized molecules, and resolution of reconstructed images based on Fourier Ring Correlation. We found that, with optimized parameters, single-molecule switching nanoscopy at high speeds can achieve the same image quality as imaging at conventional speeds in a 5-25 times shorter time period. Furthermore, we measured the photoswitching kinetics of Alexa Fluor 647 from single-molecule experiments, and, based on this kinetic data, we developed algorithms to simulate single-molecule switching nanoscopy images. We used this software tool to demonstrate how laser intensity and camera speed affect the density of active fluorophores and influence the achievable resolution. Our study provides guidelines for choosing appropriate laser intensities for imaging Alexa Fluor 647 at different speeds and a quantification protocol for future evaluations of other probes and imaging parameters.