Caged compounds are a valuable tool to photocontrol the release of enzymatic substrates in a reaction. For example, caged urea molecules were proposed to control urea delivery to urease reactions and aid the determination of the catalytic mechanism of this enzyme. Owing to the success of several caged compounds based on ruthenium complexes, we synthesized a new inorganic caged urea (RuBpy-urea) and monitored the release of free urea by infrared spectroscopy upon irradiation. Transient infrared absorption (TRIR) indicates diffusion-controlled release of urea, and kinetics studies after photolysis demonstrate the functionality of the new caged molecule for photocontrolled delivery of urea to urease. Coupling photocontrolled delivery of urea to cryo-FTIR (fourier transform infrared spectroscopy) revealed that inhibition of urease results in different FTIR spectrum than the one observed for the noninhibited urease.

[FeFe] hydrogenases are highly active catalysts for hydrogen conversion. Their active site has two components: a [4Fe-4S] electron relay covalently attached to the H-2 binding site and a diiron cluster ligated by CO, CN-, and 2-azapropane-1,3-dithiolate (ADT) ligands. Reduction of the [4Fe-4S] site was proposed to be coupled with protonation of one of its cysteine ligands. Here, we used time-resolved infrared (TRIR) spectroscopy on the [FeFe] hydrogenase from Chlamydomonas reinhardtii (CrHydA1) containing a propane-1,3-dithiolate (PDT) ligand instead of the native ADT ligand. The PDT modification does not affect the electron transfer step to [4Fe-4S](H) but prevents the enzyme from proceeding further through the catalytic cycle. We show that the rate of the first electron transfer step is independent of the pH, supporting a simple electron transfer rather than a proton-coupled event. These results have important implications for our understanding of the catalytic mechanism of [FeFe] hydrogenases and highlight the utility of TRIR.

Dihydrofolate reductase (DHFR) is a well-studied, clinically relevant enzyme known for being highly dynamic over the course of its catalytic cycle. However, the role dynamic motions play in the explicit hydride transfer from the nicotinamide cofactor to the dihydrofolate substrate remains unclear because reaction initiation and direct spectroscopic examination on the appropriate time scale for such femtosecond to picosecond motions is challenging. Here, we employ pre-steady-state kinetics to observe the hydride transfer as directly as possible in two different species of DHFR: Escherichia coli and Homo sapiens. While the hydride transfer has been well-characterized in DHFR from E. coli, improvements in time resolution now allow for sub-millisecond dead times for stopped-flow spectroscopy, which reveals that the maximum rate is indeed faster than previously recorded. The rate in the human enzyme, previously only estimated, is also able to be directly observed using cutting-edge stopped-flow instrumentation. In addition to the pH dependence of the hydride transfer rates for both enzymes, we examine the primary H/D kinetic isotope effect to reveal a temperature dependence in the human enzyme that is absent from the E. coli counterpart. This dependence, which appears above a temperature of 15 °C is a shared feature among other hydride transfer enzymes and is also consistent with computational work suggesting the presence of a fast promoting-vibration that provides donor-acceptor compression on the time scale of catalysis to facilitate the chemistry step.

The solar conversion of CO2 to low carbon fuels has been heralded as a potential solution to combat the rise in greenhouse gas emissions. Here we report the first light-driven activation of [NiFe] CODH II from Carboxydothermus hydrogenoformans for the reduction of CO2 to CO. To accomplish this, a hybrid photosystem composed of CODH II and CdSe/CdS dot-in-rod nanocrystals was developed. By incorporating a low-potential redox mediator to assist electron transfer, quantum yields up to 19% and turnover frequencies of 9 s-1 were achieved. These results represent a new standard in efficient CO2 reduction by an enzyme-based photocatalytic systems. Furthermore, successful photoactivation of CODH II allows for future exploration into the enzyme's not fully understood mechanism.

In single-molecule force spectroscopy (SMFS), a tethered molecule is stretched using a specialized instrument to study how macromolecules extend under force. One problem in SMFS is the serial and slow nature of the measurements, performed one molecule at a time. To address this long-standing challenge, we report on the origami polymer force clamp (OPFC) which enables parallelized manipulation of the mechanical forces experienced by molecules without the need for dedicated SMFS instruments or surface tethering. The OPFC positions target molecules between a rigid nanoscale DNA origami beam and a responsive polymer particle that shrinks on demand. As a proof-of-concept, we record the steady state and time-resolved mechanical unfolding dynamics of DNA hairpins using the fluorescence signal from ensembles of molecules and confirm our conclusion using modeling.

Influenza hemagglutinin is the fusion protein that mediates fusion of the viral and host membranes through a large conformational change upon acidification in the developing endosome. The "spring-loaded" model has long been used to describe the mechanism of hemagglutinin and other type 1 viral glycoproteins. This model postulates a metastable conformation of the HA2 subunit, caged from adopting a lower-free energy conformation by the HA1 subunit. Here, using a combination of biochemical and spectroscopic methods, we study a truncated construct of HA2 (HA2*, lacking the transmembrane domain) recombinantly expressed in Escherichia coli as a model for HA2 without the influence of HA1. Our data show that HA2* folds into a conformation like that of HA2 in full length HA and forms trimers. Upon acidification, HA2* undergoes a conformational change that is consistent with the change from pre- to postfusion HA2 in HA. This conformational change is fast and occurs on a time scale that is not consistent with aggregation. These results suggest that the prefusion conformation of HA2 is stable and the change to the postfusion conformation is due to protonation of HA2 itself and not merely uncaging by HA1.

We have studied the role of protein dynamics in chemical catalysis in the enzyme dihydrofolate reductase (DHFR), using a pump-probe method that employs pulsed-laser photothermal heating of a gold nanoparticle (AuNP) to directly excite a local region of the protein structure and transient absorbance to probe the effect on enzyme activity. Enzyme activity is accelerated by pulsed-laser excitation when the AuNP is attached close to a network of coupled motions in DHFR (on the FG loop, containing residues 116-132, or on a nearby alpha helix). No rate acceleration is observed when the AuNP is attached away from the network (distal mutant and His-tagged mutant) with pulsed excitation, or for any attachment site with continuous wave excitation. We interpret these results within an energy landscape model in which transient, site-specific addition of energy to the enzyme speeds up the search for reactive conformations by activating motions that facilitate this search.

Metal-ligand cooperativity is an essential feature of bioinorganic catalysis. The design principles of such cooperativity in metalloenzymes are underexplored, but are critical to understand for developing efficient catalysts designed with earth abundant metals for small molecule activation. The simple substrate requirements of reversible proton reduction by the [NiFe]-hydrogenases make them a model bioinorganic system. A highly conserved arginine residue (R355) directly above the exogenous ligand binding position of the [NiFe]-catalytic core is known to be essential for optimal function because mutation to a lysine results in lower catalytic rates. To expand on our studies of soluble hydrogenase-1 fromPyrococcus furiosus(PfSH1), we investigated the role of R355 by site-directed-mutagenesis to a lysine (R355K) using infrared and electron paramagnetic resonance spectroscopic probes sensitive to active site redox and protonation events. It was found the mutation resulted in an altered ligand binding environment at the [NiFe] centre. A key observation was destabilization of the Ni-a(3+)-C state, which contains a bridging hydride. Instead, the tautomeric Ni-a(+)-L states were observed. Overall, the results provided insight into complex metal-ligand cooperativity between the active site and protein scaffold that modulates the bridging hydride stability and the proton inventory, which should prove valuable to design principles for efficient bioinspired catalysts.

Many enzymes are known to change conformations during their catalytic cycle, but the role of these protein motions is not well understood. Escherichia coli dihydrofolate reductase (DHFR) is a small, flexible enzyme that is often used as a model system for understanding enzyme dynamics. Recently, native tryptophan fluorescence was used as a probe to study micro- to millisecond dynamics of DHFR. Yet, because DHFR has five native tryptophans, the origin of the observed conformational changes could not be assigned to a specific region within the enzyme. Here, we use DHFR mutants, each with a single tryptophan as a probe for temperature jump fluorescence spectroscopy, to further inform our understanding of DHFR dynamics. The equilibrium tryptophan fluorescence of the mutants shows that each tryptophan is in a different environment and that wild-type DHFR fluorescence is not a simple summation of all the individual tryptophan fluorescence signatures due to tryptophan–tryptophan interactions. Additionally, each mutant exhibits a two-phase relaxation profile corresponding to ligand association/dissociation convolved with associated conformational changes and a slow conformational change that is independent of ligand association and dissociation, similar to the wild-type enzyme. However, the relaxation rate of the slow phase depends on the location of the tryptophan within the enzyme, supporting the conclusion that the individual tryptophan fluorescence dynamics do not originate from a single collective motion, but instead report on local motions throughout the enzyme.

The fabrication of dynamic, transformable biomaterials that respond to environmental cues represents a significant step forward in the development of synthetic materials that rival their highly functional, natural counterparts. Here, we describe the design and synthesis of crystalline supramolecular architectures from charge-complementary heteromeric pairs of collagen-mimetic peptides (CMPs). Under appropriate conditions, CMP pairs spontaneously assemble into either 1D ultraporous (pore diameter >100 nm) tubes or 2D bilayer nanosheets due to the structural asymmetry that arises from heteromeric self-association. Crystalline collagen tubes represent a heretofore unobserved morphology of this common biomaterial. In-depth structural characterization from a suite of biophysical methods, including TEM, AFM, high-resolution cryo-EM, and SAXS/WAXS measurements, reveals that the sheet and tube assemblies possess a similar underlying lattice structure. The experimental evidence suggests that the tubular structures are a consequence of the self-scrolling of incipient 2D layers of collagen triple helices and that the scrolling direction determines the formation of two distinct structural isoforms. Furthermore, we show that nanosheets and tubes can spontaneously interconvert through manipulation of the assembly pH and systematic adjustment of the CMP sequence. Altogether, we establish initial guidelines for the construction of dynamically responsive 1D and 2D assemblies that undergo a structurally programmed morphological transition.

Oxidoreductase enzymes often perform technologically useful chemical transformations using abundant metal cofactors with high efficiency under ambient conditions. The understanding of the catalytic mechanism of these enzymes is, however, highly dependent on the availability of well-characterized and optimized time-resolved analytical techniques. We have developed an approach for rapidly injecting electrons into a catalytic system using a photoactivated nanomaterial in combination with a range of redox mediators to produce a potential jump in solution, which then initiates turnover via electron transfer (ET) to the catalyst. The ET events at the nanomaterial-mediatorcatalyst interfaces are, however, highly sensitive to the experimental conditions such as photon flux, relative concentrations of system components, and pH. Here, we present a systematic optimization of these experimental parameters for a specific catalytic system, namely, [FeFe] hydrogenase from Chlamydomonas reinhardtii (CrHydAl). The developed strategies can, however, be applied in the study of a wide variety of oxidoreductase enzymes. Our potential jump system consists of CdSe/CdS core-shell nanorods as a photosensitizer and a series of substituted bipyridinium salts as mediators with redox potentials in the range from -550 to -670 mV (vs SHE). With these components, we screened the effect of pH, mediator concentration, protein concentration, photosensitizer concentration, and photon flux on steady-state photoreduction and hydrogen production as well as ET and potential jump efficiency. By manipulating these experimental conditions, we show the potential of simple modifications to improve the tunability of the potential jump for application to study oxidoreductases.

This study reports how the length of capping ligands on a nanocrystal surface affects its interfacial electron transfer (ET) with surrounding molecular electron acceptors, and consequently, impact the H2 production of a biotic-abiotic hybrid artificial photosynthetic system. Specifically, we study how the H2 production efficiency of a hybrid system, combining CdS nanorods (NRs), [NiFe] hydrogenase, and redox mediators (propyl-bridged 2,2'-bipyridinium, PDQ2+), depends on the alkyl chain length of mercaptocarboxylate ligands on the NR surface. We observe a minor decrease of the quantum yield for H2 production from 54 ± 6 to 43 ± 2% when varying the number of methylene units in the ligands from 2 to 7. In contrast, an abrupt decrease of the yield was observed from 43 ± 2 to 4 ± 1% when further increasing n from 7 to 11. ET studies reveal that the intrinsic ET rates from the NRs to the electron acceptor PDQ2+ are all within 108-109 s-1 regardless of the length of the capping ligands. However, the number of adsorbed PDQ2+ molecules on NR surfaces decreases dramatically when n ≥ 10, with the saturating number changing from 45 ± 5 to 0.3 ± 0.1 for n = 2 and 11, respectively. These results are not consistent with the commonly perceived exponential dependence of ET rates on the ligand length. Instead, they can be explained by the change of the accessibility of NR surfaces to electron acceptors from a disordered "liquid" phase at n < 7 to a more ordered "crystalline" phases at n > ∼7. These results highlight that the order of capping ligands is an important design parameter for further constructing nanocrystal/molecular assemblies in broad nanocrystal-based applications.

Nickel-chelating lipids offer a convenient platform for reversible immobilization of histidine-tagged proteins to liposome surfaces. This interaction recently found utility as a model system for studying membrane remodeling triggered by protein crowding. Despite its wide array of utility, the molecular details of transient protein association to the lipid surfaces decorated with such chelator lipids remains poorly understood. In this study, we explore the kinetics of protein-liposome association across a wide concentration range using stopped-flow fluorescence. The fluorescence of histidine-tagged protein containing an intrinsic fluorophore (superfolder green fluorescent protein, SfGFP) was quenched upon binding to Ni-NTA-modified liposomes containing the quencher Dabsyl-PE lipids. Stopped-flow fluorescence reveals a complex, multiexponential binding behavior with a fast (kobs ∼ 10-20 s-1) phase and slower (kobs < 4 s-1) phase. Interestingly, the observed rates for the slower phase increase initially under low concentrations but start decreasing once a critical concentration is reached. Despite differences in the binding time scales, we observe that the trend of decreasing rates is reproducible irrespective of the chelator lipid doping level, protein surface charge, or lipid composition. Consideration of the protein footprint and membrane surface area occupancy leads us to conclude that the multiphasic binding behavior is reflective of protein binding via two distinct binding conformations. We propose that preliminary steps in protein association involve binding of a sterically occlusive side-on conformation followed by reorganization that leads to an end-on conformation with increased packing density. These results are important for the improvement of histidine-tag-based immobilization strategies and offer mechanistic insight into intermediates preceding membrane bending driven by protein crowding.

Hydrogenases are metalloenzymes that catalyze the reversible oxidation of H2. The [FeFe] hydrogenases are generally biased toward proton reduction and have high activities. Several different catalytic mechanisms have been proposed for the [FeFe] enzymes based on the identification of intermediate states in equilibrium and steady state experiments. Here, we examine the kinetic competency of these intermediate states in the [FeFe] hydrogenase from Chlamydomonas reinhardtii (CrHydA1), using a laser-induced potential jump and time-resolved IR (TRIR) spectroscopy. A CdSe/CdS dot-in-rod (DIR) nanocrystalline semiconductor is employed as the photosensitizer and a redox mediator efficiently transfers electrons to the enzyme. A pulsed laser induces a potential jump, and TRIR spectroscopy is used to follow the population flux through each intermediate state. The results clearly establish the kinetic competency of all intermediate populations examined: Hox, Hred, HredH+, HsredH+, and Hhyd. Additionally, a new short-lived intermediate species with a CO peak at 1896 cm-1 was identified. These results establish a kinetics framework for understanding the catalytic mechanism of [FeFe] hydrogenases.

Assemblies of inorganic nanocrystals and natural enzymes are promising systems that combine both the unique and tunable photophysical properties of nanocrystals as well as the high catalytic efficiency and selectivity of bioreaction centers. The key challenge in design of these assemblies is to favor the electron/energy transfer (ET) between the abiotic and biotic materials while retarding the undesired charge recombination. The solution to this challenge relies on both rational design of materials with proper energetic and other physical properties, and, equally important if not more so, the surface condition of each component and the configuration of their assemblies. In this study, we investigate a recently developed nanorod and NiFe hydrogenase (H2ase) assemblies which utilize a redox-mediated approach to shuttle the electron transfer between the nanorods and the H2ase enzymes with an impressive H2 production quantum yield up to 77 %.1 We study the impact of alkyl chain lengths of a common type of capping ligand for nanocrystals, mercaptoalkylcarboxylate, on the H2 production quantum yield of the system and elaborate the ligand’s impact on the underlying ET transfer between NRs and the electron acceptors. We observed the existence of an abrupt decrease of the quantum yield for H2 production of the system when increasing the alkyl chain length of the ligands from n= 7 to 10 (35 % and 12 %, respectively), whereas only a minor performance decrease is observed when n is below 7 (35 % and 42 % for n=7 and 2, respectively). These results are further shown in good agreement with the sudden decrease of the yield of the reduced mediator, propyl-bridged 2-2’-bipyridinium (PDQ2+), during the steady-state photoreduction experiments, suggesting that generation efficiency of the redox equivalents control the overall efficiency of the current redox-mediated systems. Further transient spectroscopic measurements revealed that the intrinsic ET transfer rates from the NR to the mediator PDQ are all on the order of 108 s-1 regardless of the length of the capping ligands. Instead, the amount of the average surface attached PDQ2+ molecules decreases dramatically when increasing the length of n above 7, with a saturated surface coverage of σ=40 to σ =0.5 for n=2 and n= 7, respectively. These results cannot be explained by the commonly perceived ligand length dependent ET transfer by tunneling through a barrier: k = exp (- ) on the nanorod surface, with the distance dependent constants, , reported to between 0.3 to 0.8 Å-1.2,3 Instead, these results are rationalized by the accessibility of CdS surface to electron acceptors, probably due to the change of the ligand configuration from the disordered to ordered phases. These results characterize quantitively the efficiency limiting step in the mediator based CdS nanorod/Hydrogenases assemblies, demonstrate impact of configurational arrangements of ligands on NR surface on its ET behavior, and therefore are important for the design of biotic and abiotic systems for various applications. Acknowledgment Wenxing Yang acknowledges the financial support from the Swedish Research Council for an International Postdoc Fellowship. Reference (1) Chica, B.; Wu, C. H.; Liu, Y.; Adams, M. W. W.; Lian, T.; Dyer, R. B. Balancing Electron Transfer Rate and Driving Force for Efficient Photocatalytic Hydrogen Production in CdSe/CdS Nanorod-[NiFe] Hydrogenase Assemblies. Energy Environ. Sci. 2017, 10 (10), 2245–2255. (2) Tagliazucchi, M.; Tice, D. B.; Sweeney, C. M.; Morris-Cohen, A. J.; Weiss, E. A. Ligand-Controlled Rates of Photoinduced Electron Transfer in Hybrid CdSe Nanocrystal/Poly(Viologen) Films. ACS Nano 2011, 5 (12), 9907–9917. (3) Wilker, M. B.; Utterback, J. K.; Greene, S.; Brown, K. A.; Mulder, D. W.; King, P. W.; Dukovic, G. Role of Surface-Capping Ligands in Photoexcited Electron Transfer between CdS Nanorods and [FeFe] Hydrogenase and the Subsequent H 2 Generation. J. Phys. Chem. C 2018, 122 (1), 741–750. Figure 1

A series of viologen related redox mediators of varying reduction potential has been characterized and their utility as electron shuttles between CdSe quantum dots and hydrogenase enzyme has been demonstrated. Tuning the mediator LUMO energy optimizes the performance of this hybrid photocatalytic system by balancing electron transfer rates of the shuttle.

Triosephosphate isomerase (TIM) catalyzes the interconversion between dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde 3-phosphate (GAP) via an enediol(ate) intermediate. The active-site residue Glu165 serves as the catalytic base during catalysis. It abstracts a proton from C1 carbon of DHAP to form the reaction intermediate and donates a proton to C2 carbon of the intermediate to form product GAP. Our difference Fourier transform infrared spectroscopy studies on the yeast TIM (YeTIM)/phosphate complex revealed a C═O stretch band at 1706 cm-1 from the protonated Glu165 carboxyl group at pH 7.5, indicating that the p Ka of the catalytic base is increased by >3.0 pH units upon phosphate binding, and that the Glu165 carboxyl environment in the complex is still hydrophilic in spite of the increased p Ka. Hence, the results show that the binding of the phosphodianion group is part of the activation mechanism which involves the p Ka elevation of the catalytic base Glu165. The deprotonation kinetics of Glu165 in the μs to ms time range were determined via infrared (IR) T-jump studies on the YeTIM/phosphate and ("heavy enzyme") [U-13C,-15N]YeTIM/phosphate complexes. The slower deprotonation kinetics in the ms time scale is due to phosphate dissociation modulated by the loop motion, which slows down by enzyme mass increase to show a normal heavy enzyme kinetic isotope effect (KIE) ∼1.2 (i.e., slower rate in the heavy enzyme). The faster deprotonation kinetics in the tens of μs time scale is assigned to temperature-induced p Ka decrease, while phosphate is still bound, and it shows an inverse heavy enzyme KIE ∼0.89 (faster rate in the heavy enzyme). The IR static and T-jump spectroscopy provides atomic-level resolution of the catalytic mechanism because of its ability to directly observe the bond breaking/forming process.

Functional enzyme–nanoparticle bioconjugates are increasingly important in biomedical and biotechnology applications such as drug delivery and biosensing. Optimization of the function of such bioconjugates requires careful control and characterization of their structures and activity, but current methods are inadequate for this purpose. A key shortcoming of existing approaches is the lack of an accurate method for quantitating protein content of bioconjugates for low (monolayer) surface coverages. In this study, an integrated characterization methodology for protein–gold nanoparticle (AuNP) bioconjugates is developed, with a focus on site-specific attachment and surface coverage of protein on AuNPs. Single-cysteine-containing mutants of dihydrofolate reductase are covalently attached to AuNPs with diameters of 5, 15, and 30 nm, providing a range of surface curvature. Site-specific attachment to different regions of the protein surface is investigated, including attachment to a flexible loop versus a rigid α helix. Characterization methods include SDS-PAGE, UV–vis spectrophotometry, dynamic light scattering, and a novel fluorescence-based method for accurate determination of low protein concentration on AuNPs. An accurate determination of both protein and AuNP concentration in conjugate samples allows for the calculation of the surface coverage. We find that surface coverage is related to the surface curvature of the AuNP, with a higher surface coverage observed for higher surface curvature. The combination of these characterization methods is important for understanding the functionality of protein–AuNP bioconjugates, particularly enzyme activity.

Dynamic modulation of lipid membrane curvature can be achieved by a number of peripheral protein binding mechanisms such as hydrophobic insertion of amphipathic helices and membrane scaffolding. Recently, an alternative mechanism was proposed in which crowding of peripherally bound proteins induces membrane curvature through steric pressure generated by lateral collisions. This effect was enhanced using intrinsically disordered proteins that possess high hydrodynamic radii, prompting us to explore whether membrane bending can be triggered by the folding-unfolding transition of surface-bound proteins. We utilized histidine-tagged human serum albumin bound to Ni-NTA-DGS containing liposomes as our model system to test this hypothesis. We found that reduction of the disulfide bonds in the protein resulted in unfolding of HSA, which subsequently led to membrane tubule formation. The frequency of tubule formation was found to be significantly higher when the proteins were unfolded while being localized to a phase-separated domain as opposed to randomly distributed in fluid phase liposomes, indicating that the steric pressure generated from protein unfolding can drive membrane deformation. Our results are critical for the design of peripheral membrane protein-immobilization strategies and open new avenues for exploring mechanisms of membrane bending driven by conformational changes of peripheral membrane proteins.

Membrane fusion is ubiquitous in biology, both in natural cellular functions such as neurotransmission and in pathological processes such as viral infection. The fusion of lipid bilayer membranes involves membrane contact, merger, and formation of an aqueous fusion pore, allowing the merging of separate compartments (either extracellular or intracellular) and mixing of their contents. For example, in neurotransmission, the soluble N -ethylmaleimide–sensitive factor attachment receptor (SNARE) mediates fusion of synaptic vesicles with the plasma membrane to release neurotransmitters, driven by zipping of the four-helix bundle of the SNARE complex (1). Enveloped viruses such as the influenza virus and HIV infect target host cells through an analogous protein-mediated membrane fusion process (2), releasing the viral contents into the host cell. Hence, membrane fusion is of fundamental importance and also of practical interest for its potential role in efficient drug delivery, or the prevention of viral infection by blocking the pathway of entry into the cell. Membrane fusion has significant activation barriers associated with accessing high-energy, nonbilayer intermediate structures, arising from dehydration of membrane surfaces and bending the bilayer (1, 3, 4). The barriers for the two transitions from separate membranes to a hemifused intermediate and from there to pore formation have been estimated from experiments and simulations to range from 20 to 80 k B T , yielding a total energy barrier to achieve membrane fusion of greater than 40 k B T (4). Fusion proteins may lower these barriers and provide the necessary impetus to overcome them, for example by refolding with the release of substantial free energy. Influenza hemagglutinin (HA) has served as a paradigm for understanding the mechanism of protein-mediated membrane fusion (5⇓–7), and it is also an important target for antiviral drug development (8⇓⇓⇓–12). Various mechanisms have … [↵][1]1To whom correspondence should be addressed. Email: briandyer{at}emory.edu. [1]: #xref-corresp-1-1