Double Electron-electron Resonance Research Articles

Location of the extrinsic subunit PsbP in photosystem II studied by pulsed electron-electron double resonance

The binding site of the extrinsic protein PsbP in plant photosystem II was mapped by pulsed electron-electron double resonance, using mutant spinach PsbP (Pro20Cys, Ser82Cys, Ala111Cys, and Ala186Cys) labeled with 4-maleimido-TEMPO (MSL) spin label. The distances between the spin label and the Tyr160 neutral radical (YD) in PsbD, the D2 subunit of plant photosystem II, were 50.8 ± 3.5 Å, 54.9 ± 4.0 Å, 57.8 ± 4.9 Å, and 58.4 ± 14.1 Å, respectively. The geometry inferred from these distances was fitted to the PsbP crystal structure (PDB: 4RTI) to obtain the coordinates of YD relative to PsbP. These coordinates were then fitted under boundary conditions to the structure of cyanobacterial photosystem II (PDB: 4UB6), by rotating on Euler angles centered at fixed YD coordinates. The result proposed two models which show possible acidic amino acid residues in CP43, CP47 and D2 that can bind the basic amino acids Arg48, Lys143, and Lys160 in PsbP.

Retinal guanylyl cyclase activating protein 1 forms a functional dimer.

Retinal guanylyl cyclases (RetGCs) in vertebrate photoreceptors are regulated by the guanylyl cyclase activator proteins (GCAP1 and GCAP2). Here, we report EPR double electron-electron resonance (DEER) studies on the most ubiquitous GCAP isoform, GCAP1 and site-directed mutagenesis analysis to determine an atomic resolution structural model of a GCAP1 dimer. Nitroxide spin-label probes were introduced at individual GCAP1 residues: T29C, E57C, E133C, and E154C. The intermolecular distance of each spin-label probe (measured by DEER) defined restraints for calculating the GCAP1 dimeric structure by molecular docking. The DEER-derived structural model of the GCAP1 dimer was similar within the experimental error for both the Mg2+-bound activator and Ca2+-bound inhibitor states (RMSD < 2.0 Å). The GCAP1 dimer possesses intermolecular hydrophobic contacts involving the side chain atoms of H19, Y22, F73 and V77. The structural model of the dimer was validated by GCAP1 mutations (H19R, Y22D, F73E, and V77E) at the dimer interface that each abolished protein dimerization. Previous studies have shown that each of these mutants either diminished or completely suppressed the ability of GCAP1 to activate the cyclase. These results suggest that GCAP1 dimerization may affect compartmentalization of GCAP1 in the photoreceptors and/or affect regulation of the cyclase activity.

Conformational transitions of the sodium-dependent sugar transporter, vSGLT

Sodium-dependent transporters couple the flow of Na+ ions down their electrochemical potential gradient to the uphill transport of various ligands. Many of these transporters share a common core structure composed of a five-helix inverted repeat and deliver their cargo utilizing an alternating-access mechanism. A detailed characterization of inward-facing conformations of the Na+-dependent sugar transporter from Vibrio parahaemolyticus (vSGLT) has previously been reported, but structural details on additional conformations and on how Na+ and ligand influence the equilibrium between other states remains unknown. Here, double electron-electron resonance spectroscopy, structural modeling, and molecular dynamics are utilized to deduce ligand-dependent equilibria shifts of vSGLT in micelles. In the absence and presence of saturating amounts of Na+, vSGLT favors an inward-facing conformation. Upon binding both Na+ and sugar, the equilibrium shifts toward either an outward-facing or occluded conformation. While Na+ alone does not stabilize the outward-facing state, gating charge calculations together with a kinetic model of transport suggest that the resting negative membrane potential of the cell, absent in detergent-solubilized samples, may stabilize vSGLT in an outward-open conformation where it is poised for binding external sugars. In total, these findings provide insights into ligand-induced conformational selection and delineate the transport cycle of vSGLT.

Inhibitor-Directed Spin Labelling-A High Precision and Minimally Invasive Technique to Study the Conformation of Proteins in Solution.

Pulsed electron-electron double resonance spectroscopy (known as PELDOR or DEER) has recently become a very popular tool in structural biology. The technique can be used to accurately measure distance distributions within macromolecules or macromolecular complexes, and has become a standard method to validate structural models and to study the conformational flexibility of macromolecules. It can be applied in solution, in lipid environments or even in cells. Because most biological macromolecules are diamagnetic, they are normally invisible for PELDOR spectroscopy. To render a particular target molecule accessible for PELDOR, it can be engineered to contain only one or two surface-exposed cysteine residues, which can be efficiently spin-labelled using thiol-reactive nitroxide compounds. This method has been coined "site-directed spin labelling" (SDSL) and is normally straight-forward. But, SDSL can be very challenging for proteins with many native cysteines, or even a single functionally or structurally important cysteine residue. For such cases, alternative spin labelling techniques are needed. Here we describe the concept of "inhibitor-directed spin labelling" (IDSL) as an approach to spin label suitable cysteine-rich proteins in a site-directed and highly specific manner by employing bespoke spin-labelled inhibitors. Advantages and disadvantages of IDSL are discussed.

Studying structure and function of membrane proteins with PELDOR/DEER spectroscopy - The crystallographers' perspective.

In 1985, the first X-ray structure of a membrane protein was determined. Today, more than 30 years later, many more structures have been solved. Nevertheless, studying the structure of membrane proteins remains a very challenging task. Due to their inherent conformational flexibility, having a single X-ray structure is usually only the first step towards truly understanding the function of these dynamic molecules. For this reason, additional methods are needed that can provide complementary information, especially about conformational flexibility. Pulsed electron-electron double resonance spectroscopy (PELDOR, also known as DEER) is such a method. It can be used to precisely measure nanometer distance distributions between intrinsic or artificially introduced spin-centers in macromolecules and thereby to probe the conformational state of the macromolecule. PELDOR can be applied in solution, in detergent, in lipid bilayers and even within cells. However, PELDOR is an advanced spectroscopy technique and requires specialised equipment and training. This chapter aims to be a starting point for crystallographers and other structural biologists who want to get a better understanding of PELDOR spectroscopy and its application. It gives an insight into the planning stages of the experiment (i.e., which spin labels are possible and where to place them), how a PELDOR experiment is conducted and how the results are interpreted. For this purpose, the substrate binding protein (SBP) from a Vibrio cholerae TRAP transporter is used as a step-by-step example. Further, the chapter gives examples of how PELDOR spectroscopy has previously been applied to overcome known limitations of X-ray crystallography in modern integrative structural biology approaches.

Chirp echo Fourier transform EPR-detected NMR

A new ultra-wide band (UWB) pulse EPR method is introduced for observing all nuclear frequencies of a paramagnetic center in a single shot. It is based on burning spectral holes with a high turning angle (HTA) pulse that excites forbidden transitions and subsequent detection of the hole pattern by a chirp echo. We term this method Chirp Echo Epr SpectroscopY (CHEESY)-detected NMR. The approach is a revival of FT EPR-detected NMR. It yields similar spectra and the same type of information as electron-electron double resonance (ELDOR)-detected NMR, but with a multiplex advantage. We apply CHEESY-detected NMR in Q band to nitroxides and correlate the hyperfine spectrum to the EPR spectrum by varying the frequency of the HTA pulse. Furthermore, a selective π pulse before the HTA pulse allows for detecting hyperfine sublevel correlations between transitions of one nucleus and for elucidating the coupling regime, the same information as revealed by the HYSCORE experiment. This is demonstrated on hexaaquamanganese(II). We expect that CHEESY-detected NMR is generally applicable to disordered systems and that our results further motivate the development of EPR spectrometers capable of coherent UWB excitation and detection, especially at higher fields and frequencies.

A Structural Model of a P450-Ferredoxin Complex from Orientation-Selective Double Electron-Electron Resonance Spectroscopy.

Cytochrome P450 (CYP) monooxygenases catalyze the oxidation of chemically inert carbon-hydrogen bonds in diverse endogenous and exogenous organic compounds by atmospheric oxygen. This C-H bond oxy-functionalization activity has huge potential in biotechnological applications. Class I CYPs receive the two electrons required for oxygen activation from NAD(P)H via a ferredoxin reductase and ferredoxin. The interaction of Class I CYPs with their cognate ferredoxin is specific. In order to reconstitute the activity of diverse CYPs, structural characterization of CYP-ferredoxin complexes is necessary, but little structural information is available. Here we report a structural model of such a complex (CYP199A2-HaPux) in frozen solution derived from distance and orientation restraints gathered by the EPR technique of orientation-selective double electron-electron resonance (os-DEER). The long-lived oscillations in the os-DEER spectra were well modeled by a single orientation of the CYP199A2-HaPux complex. The structure is different from the two known Class I CYP-Fdx structures: CYP11A1-Adx and CYP101A1-Pdx. At the protein interface, HaPux residues in the [Fe2S2] cluster-binding loop and the α3 helix and the C-terminus residue interact with CYP199A2 residues in the proximal loop and the C helix. These residue contacts are consistent with biochemical data on CYP199A2-ferredoxin binding and electron transfer. Electron-tunneling calculations indicate an efficient electron-transfer pathway from the [Fe2S2] cluster to the heme. This new structural model of a CYP-Fdx complex provides the basis for tailoring CYP enzymes for which the cognate ferredoxin is not known, to accept electrons from HaPux and display monooxygenase activity.

Spin properties of dense near-surface ensembles of nitrogen-vacancy centers in diamond

We present a study of the spin properties of dense layers of near-surface nitrogen-vacancy (NV) centres in diamond created by nitrogen ion implantation. The optically detected magnetic resonance contrast and linewidth, spin coherence time, and spin relaxation time, are measured as a function of implantation energy, dose, annealing temperature and surface treatment. To track the presence of damage and surface-related spin defects, we perform in situ electron spin resonance spectroscopy through both double electron-electron resonance and cross-relaxation spectroscopy on the NV centres. We find that, for the energy ($4-30$~keV) and dose ($5\times10^{11}-10^{13}$~ions/cm$^2$) ranges considered, the NV spin properties are mainly governed by the dose via residual implantation-induced paramagnetic defects, but that the resulting magnetic sensitivity is essentially independent of both dose and energy. We then show that the magnetic sensitivity is significantly improved by high-temperature annealing at $\geq1100^\circ$C. Moreover, the spin properties are not significantly affected by oxygen annealing, apart from the spin relaxation time, which is dramatically decreased. Finally, the average NV depth is determined by nuclear magnetic resonance measurements, giving $\approx10$-17~nm at 4-6 keV implantation energy. This study sheds light on the optimal conditions to create dense layers of near-surface NV centres for high-sensitivity sensing and imaging applications.

Optimal Tikhonov regularization for DEER spectroscopy

Tikhonov regularization is the most commonly used method for extracting distance distributions from experimental double electron-electron resonance (DEER) spectroscopy data. This method requires the selection of a regularization parameter, α, and a regularization operator, L. We analyze the performance of a large set of α selection methods and several regularization operators, using a test set of over half a million synthetic noisy DEER traces. These are generated from distance distributions obtained from in silico double labeling of a protein crystal structure of T4 lysozyme with the spin label MTSSL. We compare the methods and operators based on their ability to recover the model distance distributions from the noisy time traces. The results indicate that several α selection methods perform quite well, among them the Akaike information criterion and the generalized cross validation method with either the first- or second-derivative operator. They perform significantly better than currently utilized L-curve methods.

Identifying the Optimal Level of Regularization in Deer Data Analysis

Double Electron-Electron Resonance (DEER) spectroscopy is an Electron Paramagnetic Resonance (EPR) experiment that accurately reports the distribution of distances, P(r), between two or more unpaired electrons over the range of about 10 to 160 ångströms. DEER is becoming increasingly important as a tool for structural biology due to its ability to elucidate protein conformational landscapes, as well as its amenability to complex samples such as membrane-bound proteins and large protein complexes. The spin-spin distance distribution, P(r), is most often inferred from the time-domain experimental data with a regularized least-squares method known as Tikhonov regularization. Although this method typically provides reliable estimates of P(r), it requires the input of a regularization parameter, α, which scales the magnitude of a penalty term with respect to the fitting error. This regularization parameter significantly affects the estimated P(r), so selecting its optimal value is crucial for accurate analysis of DEER data. The most popular procedure for selecting α in DEER data analysis is a graphical method called the L-curve criterion, which has empirical justification. However, to date, a systematic exploration of the many extant regularization parameter selection methods has not been carried out in the context of DEER data analysis. Here, we evaluate 16 methods, including the L-curve criterion, against a large, physically-derived test set of simulated DEER data. The test set of P(r)s was generated using a side-chain rotamer library in conjunction with a crystal structure (2LZM). This test set was then used to generate a large set of experimentally realistic simulated time-domain data.

The Conformation of Alpha-Synuclein on the Natural Membrane Mimics Inner Mitochondrial Membrane (IMM) and Neuronal Plasma Membrane (NPM) Revisited by Bayesian Analysis of Double Electron Electron Resonance (DEER) Distance Distributions

The Conformation of Alpha-Synuclein on the Natural Membrane Mimics Inner Mitochondrial Membrane (IMM) and Neuronal Plasma Membrane (NPM) Revisited by Bayesian Analysis of Double Electron Electron Resonance (DEER) Distance Distributions

Effects of Natural Polymorphisms of non-B HIV-1 Protease on Protein Conformations

HIV-1 protease (PR) is essential to the maturation of HIV-1 virus as it post-translationally cleaves the viral polyproteins gag and gag-pol. Inhibition of HIV-1 PR lead to non-infectious immature virus. This makes HIV-1 PR a drug target for HIV infection. The flaps of HIV-1 PR play an important role in its catalytic activity as they control access of substrate as well as inhibitor to the catalytic pocket of the protease. The flaps of HIV-1 PR adopt different conformations: closed, semi-open, wide-open, and curled-open [1]. Our previous study suggests natural polymorphisms can affect conformational ensembles of the protease [2]. In this study, double electron-electron resonance (DEER) is utilized to study how natural polymorphisms affect protein conformational ensembles in non-B HIV-1 PR variants including subtypes D, H, and F, and CRFs AC, AG, and BF. Most of these variants showed conformational ensembles similar to that of multidrug resistant variant. Darunavir, the most potent protease inhibitor to date, was unable to shift flap conformation of CRF_AC to closed conformation which is indicative of drug resistance.

Probing Conformational Motions Underlying Anesthetic Drug Actions in a Ligand-Gated Ion Channel

General anesthetics and ethanol exert their functional effects on pentameric ligand-gated ion channels (pLGICs) by binding to distinct allosteric sites that are far from the agonist binding sites. While recent high-resolution structures of prokaryotic and eukaryotic pLGICs, solved in the presence of these drugs have illuminated their binding sites, the structural mechanisms underlying how these drugs enhance or inhibit pLGIC function are largely unknown. Recently, a single residue variant in the bacterial pLGIC, GLIC, was identified that is potentiated by ethanol (PNAS 108:12149-12154, 2011). Taking advantage of this GLIC ethanol-sensitive mutant, we are using site directed spin labeling electron paramagnetic resonance (SDSL EPR) spectroscopy to study motions underlying ethanol potentiation of GLIC currents. Previous studies have shown that agonist-mediated channel opening is associated with an inward tilting of the extracellular binding domain (ECD). Here, we examined if, and how, the ECD moves in response to ethanol binding in the transmembrane channel domain. Individual cysteine residues were introduced in the ECD of an ethanol-sensitive GLIC construct (F14’A) at the top of β-strands (β1, 2, 5, 6, 8), labeled with the spin probe MTSL and ethanol-induced motions were measured using double electron-electron resonance (DEER) spectroscopy and compared to agonist (proton)-induced gating motions. DEER distance distributions for spin labels attached to N19C (β1), S93C (β5) and Q101C (β6) revealed that the attached probes moved closer together at pH 3.0, indicating that the inner β-sheet of GLIC ECD tilts inwards upon agonist activation. Interestingly, DEER distance distributions for N19 (β1) and S93 (β5) showed no significant changes in the presence of ethanol, indicating that structural mechanisms underlying ethanol allosteric drug modulation of pLGIC function are different than agonist-mediated motions that lead to channel activation.

Expression and Purification of the Pain Receptor TRPV1 for Spectroscopic Analysis

The transient receptor potential vanilloid 1 (TRPV1) channel is an essential component of the cellular mechanism through which noxious stimuli evoke pain. Functional and structural characterizations of TRPV1 shed light on vanilloid activation, yet the mechanisms for temperature and proton gating remain largely unknown. Spectroscopic approaches are needed to understand the mechanisms by which TRPV1 translates diverse stimuli into channel opening. Here, we have engineered a minimal cysteine-less rat TRPV1 construct (eTRPV1) that can be stably purified and reconstituted for spectroscopic studies. Biophysical analyses of TRPV1 constructs reveal that the S5-pore helix loop influences protein stability and vanilloid and proton responses, but not thermal sensitivity. Cysteine mutants retain function and stability for double electron-electron resonance (DEER) and electron paramagnetic resonance (EPR) spectroscopies. DEER measurements in the closed state demonstrate that eTRPV1 reports distances in the extracellular vestibule, equivalent to those observed in the apo TRPV1 structure. EPR measurements show a distinct pattern of mobilities and spectral features, in detergent and liposomes, for residues at the pore domain that agree with their location in the TRPV1 structure. Our results set the stage for a systematic characterization of TRPV1 using spectroscopic approaches to reveal conformational changes compatible with thermal- and ligand-dependent gating.

Capturing Conformational Changes of the Tau Protein Upon Aggregation

Many neurodegenerative diseases (NDs) are associated with abnormal accumulation of protein aggregates in the brain. The intrinsically disordered tau protein, which does not possess a well-defined 3D structure in normal conditions, is involved in several NDs, including Alzheimer diseases and frontotemporal dementia. In pathological conditions, tau can aggregate into structured aggregates, in so-called neurofibrillarly tangles, which are deadly to neurons. Different fibers supra-structures, referred to as strains, have been shown to be associated with different diseases, thereby suggesting that the arrangement of tau molecules in the fibers is pathologically relevant. Strikingly, tau has recently been termed as a prion-like protein because of the capacity of one tau strains to effectively propagate from neuron to neuron. The ability of a strain to convert naïve disordered monomers into specific structured aggregates is still not understood, but conformational changes of tau is thought to be a critical factor. Here we have characterized tau structure by combining electron paramagnetic resonance spectroscopy and spin-labelling techniques. In particular, double electron-electron resonance spectroscopy (DEER) allowed us to measure intra-molecular distances at different time points along the aggregation process, in particular at early times before the formation of beta-sheets structures. We have used different aggregation triggers including heparin cofactors and fiber seeds extracted from model mouse brains. We will show some key conformational changes occurring during aggregation, and relate them to the seeding capacity of tau misfolded species. A better view of tau disorder-to-structure transition will help understand the aggregation mechanisms, and pave the way to new drug development strategies that aim at limiting propagation of deleterious aggregates in NDs.

Coiled-coil formation of the membrane-fusion K/E peptides viewed by electron paramagnetic resonance.

The interaction of the complementary K (Ac-(KIAALKE)3-GW-NH2) and E (Ac-(EIAALEK)3-GY-NH2) peptides, components of the zipper of an artificial membrane fusion system (Robson Marsden H. et al. Angew Chemie Int Ed. 2009) is investigated by electron paramagnetic resonance (EPR). By frozen solution continuous-wave EPR and double electron-electron resonance (DEER), the distance between spin labels attached to the K- and to the E-peptide is measured. Three constructs of spin-labelled K- and E-peptides are used in five combinations for low temperature investigations. The K/E heterodimers are found to be parallel, in agreement with previous studies. Also, K homodimers in parallel orientation were observed, a finding that was not reported before. Comparison to room-temperature, solution EPR shows that the latter method is less specific to detect this peptide-peptide interaction. Combining frozen solution cw-EPR for short distances (1.8 nm to 2.0 nm) and DEER for longer distances thus proves versatile to detect the zipper interaction in membrane fusion. As the methodology can be applied to membrane samples, the approach presented suggests itself for in-situ studies of the complete membrane fusion process, opening up new avenues for the study of membrane fusion.

Chain Assembly and Disassembly Processes Differently Affect the Conformational Space of Ubiquitin Chains

Ubiquitination is the most versatile posttranslational modification. The information is encoded by linkage type as well as chain length, which are translated by ubiquitin binding domains into specific signaling events. Chain topology determines the conformational space of a ubiquitin chain and adds an additional regulatory layer to this ubiquitin code. In particular, processes that modify chain length will be affected by chain conformations as they require access to the elongation or cleavage sites. We investigated conformational distributions in the context of chain elongation and disassembly using pulsed electron-electron double resonance spectroscopy in combination with molecular modeling. Analysis of the conformational space of diubiquitin revealed conformational selection or remodeling as mechanisms for chain recognition during elongation or hydrolysis, respectively. Chain elongation to tetraubiquitin increases the sampled conformational space, suggesting that a high intrinsic flexibility of K48-linked chains may contribute to efficient proteasomal degradation.

Conformational Coupling and trans-Inhibition in the Human Antigen Transporter Ortholog TmrAB Resolved with Dipolar EPR Spectroscopy.

ATP-binding cassette (ABC) exporters actively move chemically diverse substrates across biological membranes. Their malfunction leads to human diseases. Many ABC exporters encompass asymmetric nucleotide-binding sites (NBSs), and some of them are inhibited by the transported substrate. The functional relevance of the catalytic asymmetry or the mechanism for trans-inhibition remains elusive. Here, we investigated TmrAB, a functional homologue of the human antigen translocation complex TAP using advanced electron-electron double resonance spectroscopy. In the presence of ATP, the heterodimeric ABC exporter exists in a tunable equilibrium between inward- and outward-facing conformations. The two NBSs exhibit pronounced asymmetry in the open-to-close equilibrium. The closed conformation is more favored at the degenerate NBS, and closure of either of the NBS is sufficient to open the extracellular gate. We define the mechanistic basis for trans-inhibition, which operates by a reverse transition from the outward-facing state through an occluded conformation. These novel findings uncover the central role of reversible conformational equilibrium in the function and regulation of an ABC exporter and establish a mechanistic framework for future investigations on other medically important transporters with imprinted asymmetry. Also, this study demonstrates for the first-time the feasibility to resolve equilibrium populations at multiple domains and their interdependence for global conformational changes in a large membrane protein complex.

Identification of a ubiquitin-binding interface using Rosetta and DEER

ExoU is a type III-secreted cytotoxin expressing A2 phospholipase activity when injected into eukaryotic target cells by the bacterium Pseudomonas aeruginosa The enzymatic activity of ExoU is undetectable in vitro unless ubiquitin, a required cofactor, is added to the reaction. The role of ubiquitin in facilitating ExoU enzymatic activity is poorly understood but of significance for designing inhibitors to prevent tissue injury during infections with strains of P. aeruginosa producing this toxin. Most ubiquitin-binding proteins, including ExoU, demonstrate a low (micromolar) affinity for monoubiquitin (monoUb). Additionally, ExoU is a large and dynamic protein, limiting the applicability of traditional structural techniques such as NMR and X-ray crystallography to define this protein-protein interaction. Recent advancements in computational methods, however, have allowed high-resolution protein modeling using sparse data. In this study, we combine double electron-electron resonance (DEER) spectroscopy and Rosetta modeling to identify potential binding interfaces of ExoU and monoUb. The lowest-energy scoring model was tested using biochemical, biophysical, and biological techniques. To verify the binding interface, Rosetta was used to design a panel of mutations to modulate binding, including one variant with enhanced binding affinity. Our analyses show the utility of computational modeling when combined with sensitive biological assays and biophysical approaches that are exquisitely suited for large dynamic proteins.

Small neutral Gd(iii) tags for distance measurements in proteins by double electron-electron resonance experiments.

Spin labels containing a Gd(iii) ion have become important for measuring nanometer distances in proteins by double electron-electron resonance (DEER) experiments at high EPR frequencies. The distance resolution and sensitivity of these measurements strongly depend on the Gd(iii) tag used. Here we report the performance of two Gd(iii) tags, propargyl-DO3A and C11 in DEER experiments carried out at W-band (95 GHz). Both tags are small, uncharged and devoid of bulky hydrophobic pendants. The propargyl-DO3A tag is designed for conjugation to the azide-group of an unnatural amino acid. The C11 tag is a new tag designed for attachment to a single cysteine residue. The tags delivered narrower distance distributions in the E. coli aspartate/glutamate binding protein and the Zika virus NS2B-NS3 protease than previously established Gd(iii) tags. The improved performance is consistent with the absence of specific hydrophobic or charge-charge interactions with the protein. In the case of the Zika virus NS2B-NS3 protease, unexpectedly broad Gd(iii)-Gd(iii) distance distributions observed with the previously published charged C9 tag, but not the C11 tag, illustrate the potential of tags to perturb a labile protein structure and the importance of different tags. The results obtained with the C11 tag demonstrate the closed conformation in the commonly used linked construct of the Zika virus NS2B-NS3 protease, both in the presence and absence of an inhibitor.