Slow Conformational Change Research Articles

Trapping a non-cognate nucleotide upon initial binding for replication fidelity control in SARS-CoV-2 RNA dependent RNA polymerase.

The RNA dependent RNA polymerase (RdRp) in SARS-CoV-2 is a highly conserved enzyme responsible for viral genome replication/transcription. To understand how the viral RdRp achieves fidelity control during such processes, here we computationally investigate the natural non-cognate vs. cognate nucleotide addition and selectivity during viral RdRp elongation. We focus on the nucleotide substrate initial binding (RdRp active site open) to the prechemical insertion (active site closed) of the RdRp. The current studies were first carried out using microsecond ensemble equilibrium all-atom molecular dynamics (MD) simulations. Due to the slow conformational changes (from open to closed) during nucleotide insertion and selection, enhanced or umbrella sampling methods have been further employed to calculate the free energy profiles of the nucleotide insertion. Our studies find notable stability of noncognate dATP and GTP upon initial binding in the active-site open state. The results indicate that while natural cognate ATP and Remdesivir drug analogue (RDV-TP) are biased toward stabilization in the closed state to facilitate insertion, the natural non-cognate dATP and GTP can be well trapped in off-path initial binding configurations and prevented from insertion so that to be further rejected. The current work thus presents the intrinsic nucleotide selectivity of SARS-CoV-2 RdRp for natural substrate fidelity control, which should be considered in antiviral drug design.

Influence of Atmospheric Contaminants on the Work Function of Graphite.

Airborne hydrocarbon contamination occurs rapidly on graphitic surfaces and negatively impact many of their material properties, yet much of the molecular details of the contamination remains unknown. We use Kelvin probe force microscopy (KPFM) to study the time evolution of the surface potential of graphite exposed to ambient. After exfoliation in air, the surface potential of graphite is not homogeneous and contains features that are absent in the topography image. In addition, the heterogeneity of the surface potential images increased in the first few days followed by a decrease at longer exposure times. These observations are strong support of slow conformation change, phase separation, and/or dynamic displacement of the adsorbed airborne contaminants.

Slow conformational changes in the rigid and highly stable chymotrypsin inhibitor 2.

Slow conformational changes are often directly linked to protein function. It is however less clear how such processes may perturb the overall folding stability of a protein. We previously found that the stabilizing double mutant L49I/I57V in the small protein chymotrypsin inhibitor 2 from barley led to distributed increased nanosecond and faster dynamics. Here we asked what effects the L49I and I57V substitutions, either individually or together, have on the slow conformational dynamics of CI2. We used 15 N CPMG spin relaxation dispersion experiments to measure the kinetics, thermodynamics and structural changes associated with slow conformational change in CI2. These changes result in an excited state that is populated to 4.3% at 1 °C. As the temperature is increased the population of the excited state decreases. Structural changes in the excited state are associated with residues that interact with water molecules that have well defined positions and are found at these positions in all crystal structures of CI2. The substitutions in CI2 have only little effect on the structure of the excited state whereas the stability of the excited state to some extent follows the stability of the main state. The minor state is thus most populated for the most stable CI2 variant and least populated for the least stable variant. We hypothesize that the interactions between the substituted residues and the well-ordered water molecules links subtle structural changes around the substituted residues to the region in the protein that experience slow conformational changes. This article is protected by copyright. All rights reserved.

Non‐Competitive Inhibition by a Substrate Mimetic of Human 5‐Lipoxygenase

Human 5‐lipoxygenase (5‐LOX) initiates the synthesis of the leukotrienes (LT) through a two‐step reaction: addition of molecular oxygen to arachidonic acid (AA) into 5–hydroperoxyeicosatetraenoic acid (5‐HPETE) and hydrolysis of 5‐HPETE into leukotriene A4. LTs are highly potent, pro‐inflammatory mediators, which have been implicated in asthmatic and allergic responses. 5‐LOX is mobilized in leukocytes by Ca2+ binding and targets to the membrane to receive substrate from its helper protein, 5‐lipoxygenase‐activating protein (FLAP). These allosteric partners are unnecessary in in vitro experiments but enhance activity in intact cells. The crystal structure of 5‐LOX was revealed in a “closed conformation” with the catalytic iron inaccessible to surface solvent. This closed active site is the result of a unique conformation of helix alpha 2 (Hα2) where two aromatic residues plug the putative substrate‐access portal. 5‐LOX has a disjointed Hα2 of 3 turns connected with multiple loop‐to‐helix transitions permitting this plugged conformation of the active site. For comparison, the structure of another human isoform, 15‐LOX‐2, has Hα2 in a fully elongated conformation of 6 turns and the catalytic iron solvent accessible for substrate entry. The detergent C8E4 was co‐crystallized with 15‐LOX‐2 in the active site, kinetically identified as a competitive inhibitor, and is an excellent substrate mimetic of AA. Initial kinetic studies of 5‐LOX with C8E4 have shown non‐competitive inhibition patterns. We detailed a non‐Michaelis‐Menton kinetic response of 5‐LOX to increasing amounts of substrate at the last EB meeting (https://doi.org/10.1096/fasebj.2020.34.s1.01876). We hypothesized that a ligand‐induced slow transition of the conformation of Hα2 causes the sigmoidal response to substrate by the monomeric enzyme. Kinetic cooperativity of monomeric enzymes with single substrate‐binding sites, while rare, is represented in highly regulated enzymes. We wanted to further elaborate on this model by performing inhibition kinetics of a substrate mimetic of AA to 5‐LOX. Non‐competitive inhibition of an active site binder is further evidence of a slow conformational change, as it indicates a two‐step inhibition mechanism with the slow transition to an inactive enzyme state. Understanding the inhibition kinetics of an enzyme that adopts multiple conformations and detailing the molecular determinants responsible for inhibitor binding could help delineate some of the discrepancies observed for different 5‐LOX inhibitor potencies in different cellular environments. Anti‐leukotriene therapies has proven to be a successful avenue for lowering inflammation in many disease states and more informed structure‐based drug design approach could hasten their development.

Slow Conformational Changes of Blue Light Sensor BLUF Proteins in Milliseconds.

Blue light sensor using flavin (BLUF) proteins consist of flavin-binding BLUF domains and functional domains. Upon blue light excitation, the hydrogen bond network around the flavin chromophore changes, and the absorption spectrum in the visible region exhibits a red shift. Ultimately, the light information received in the BLUF domain is transmitted to the functional region. It has been believed that this red shift is complete within nanoseconds. In this study, slow reaction kinetics were discovered in milliseconds (τ1- and τ2-phase) for all the BLUF proteins examined (AppA, OaPAC, BlrP1, YcgF, PapB, SyPixD, and TePixD). Despite extensive reports on BLUF, this is the first clear observation of the BLUF protein absorption change with the duration in the millisecond time region. From the measurements of some domain-deleted mutants of OaPAC and two chimeric mutants of PixD proteins, it was found that the slower dynamics (τ2-phase) are strongly affected by the size and nature of the C-terminal region adjacent to the BLUF domain. Hence, this millisecond reaction is a significant indicator of conformational changes in the C-terminal region, which is essential for the biological functions. On the other hand, the τ1-phase commonly exists in all BLUF proteins, including any mutants. The origin of the slow dynamics was studied using site-specific mutants. These results clearly show the importance of Trp in the BLUF domain. Based on this, a reaction scheme for the BLUF reaction is proposed.

Structural mechanism underlying ligand binding and activation of PPARγ

Ligands bind to an occluded orthosteric ligand-binding pocket within the nuclear receptor ligand-binding domain. Molecular simulations have revealed theoretical ligand entry/exit pathways to the orthosteric pocket; however, it remains unclear whether ligand binding proceeds through induced fit or conformational selection mechanisms. Here, using nuclear magnetic resonance spectroscopy, isothermal titration calorimetry, and surface plasmon resonance analysis, we provide evidence that structurally distinct agonists bind peroxisome proliferator-activated receptor γ (PPARγ) via a two-step induced fit mechanism involving an initial fast kinetic step followed by a slow conformational change. The agonist encounter complex binding pose is suggested in crystal structures where ligands bind to a surface pore suggested as a ligand entry site in molecular simulations. Our findings suggest an activation mechanism for PPARγ whereby agonist binding occurs through an initial encounter complex followed by a transition of the ligand into the final binding pose within the orthosteric pocket, inducing a transcriptionally active conformation.

Single-Molecule Fluorescence-Based Measurements of Conformational Dynamics of Calcium-Binding Protein Recoverin

Recoverin is a calcium-binding protein expressed primarily in the photoreceptor cells of the vertebrate retina, where it participates in the regulation of phototransduction and adaptation of the visual system to background light. Binding of calcium ions to recoverin induces large conformational changes, including the release of its post-translationally attached myristoyl group from a hydrophobic cavity of the protein into the solution. Previously, it was shown that the fluorophore Alexa647 site-specifically bound to a unique cysteine-39 of recoverin can be employed as a reporter of its Ca2+-dependent conformational transitions since these transitions affect fluorescence lifetime, and spectra of the dye. Here, we addressed a question, whether the Ca2+-dependent conformers of recoverin, previously resolved by NMR, can be recorded using single-molecule fluorescence microscopy. To this end, the samples of recombinant bovine recoverin site-specifically labeled at cysteine-39 with either Alexa647 or sulfo-Cy5 maleimide-functionalized fluorophores were prepared. Both forms of the protein exhibited Ca2+-induced conformational changes as revealed by steady-state spectrometry of intrinsic tryptophans fluorescence. Interestingly, two chemically similar cyanine dyes (Alexa647 and sulfo-Cy5) prodruced quite different effects on recoverin state and exhibited different photophysical responses to changes in its conformation. We performed single-molecule experiments with recoverin fluorescently labeled with Alexa647. Using a confocal setup with multiparameter fluorescence detection (MFD) we measured fluorescence lifetimes and anisotropies of bursts from individual recoverin molecules. Observation of individual molecules in solution is limited by their diffusion time (typically, 1-10 ms) - to track slower conformational changes we specifically immobilized recoverin using a biotin-streptavidin tether. Using a TIRF microscope we measured intensity traces from individual immobilized molecules. Our results show that conformational dynamics of recoverin can be measured at the single-molecule level using the protein site-specifically labeled at cysteine-39 with Alexa647. This work is supported by RFBR (№20-34-70034).

Anomalously Slow Conformational Change Dynamics of Polar Groups Anchored to Hydrophobic Surfaces in Aqueous Media.

Water molecules within a thin hydration layer, spontaneously generated on hydrophobic protein surfaces, are reported to form a poorly dynamic network structure. However, how such a water network affects the conformational change dynamics of polar groups has never been explored, although such polar groups play a critical role in protein-protein and protein-ligand interactions. In the present work, we utilized as model protein surfaces a series of self-assembled monolayers (SAMs) appended with polar (Fmoc) or ionic (FITC) fluorescent head groups that were tethered via a 1.5-nm-long flexible oligoether chain to a hydrophobic silicon wafer surface, which was densely covered with paraffinic chains. We found that, not only in deionized water but also in aqueous buffer, these oligoether-appended head groups at ambient temperatures both displayed an anomalously slow conformational change, which required ∼10 h to reach a thermodynamically equilibrated state. We suppose that these behaviors reflect the poorly dynamic and low-permittivity natures of the thin hydration layer.

Site-Specific Tryptophan Labels Reveal Local Microsecond-Millisecond Motions of Dihydrofolate Reductase.

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.

Conformational heterogeneity of Savinase from NMR, HDX-MS and X-ray diffraction analysis.

BackgroundSeveral examples have emerged of enzymes where slow conformational changes are of key importance for function and where low populated conformations in the resting enzyme resemble the conformations of intermediate states in the catalytic process. Previous work on the subtilisin protease, Savinase, from Bacillus lentus by NMR spectroscopy suggested that this enzyme undergoes slow conformational dynamics around the substrate binding site. However, the functional importance of such dynamics is unknown.MethodsHere we have probed the conformational heterogeneity in Savinase by following the temperature dependent chemical shift changes. In addition, we have measured changes in the local stability of the enzyme when the inhibitor phenylmethylsulfonyl fluoride is bound using hydrogen-deuterium exchange mass spectrometry (HDX-MS). Finally, we have used X-ray crystallography to compare electron densities collected at cryogenic and ambient temperatures and searched for possible low populated alternative conformations in the crystals.ResultsThe NMR temperature titration shows that Savinase is most flexible around the active site, but no distinct alternative states could be identified. The HDX shows that modification of Savinase with inhibitor has very little impact on the stability of hydrogen bonds and solvent accessibility of the backbone. The most pronounced structural heterogeneities detected in the diffraction data are limited to alternative side-chain rotamers and a short peptide segment that has an alternative main-chain conformation in the crystal at cryo conditions. Collectively, our data show that there is very little structural heterogeneity in the resting state of Savinase and hence that Savinase does not rely on conformational selection to drive the catalytic process.

Backmapping triangulated surfaces to coarse-grained membrane models

Many biological processes involve large-scale changes in membrane shape. Computer simulations of these processes are challenging since they occur across a wide range of spatiotemporal scales that cannot be investigated in full by any single current simulation technique. A potential solution is to combine different levels of resolution through a multiscale scheme. Here, we present a multiscale algorithm that backmaps a continuum membrane model represented as a dynamically triangulated surface (DTS) to its corresponding molecular model based on the coarse-grained (CG) Martini force field. Thus, we can use DTS simulations to equilibrate slow large-scale membrane conformational changes and then explore the local properties at CG resolution. We demonstrate the power of our method by backmapping a vesicular bud induced by binding of Shiga toxin and by transforming the membranes of an entire mitochondrion to near-atomic resolution. Our approach opens the way to whole cell simulations at molecular detail.

Probing the Conformational Changes Associated with DNA Binding to PARP1.

Poly(ADP-ribose) polymerase 1 (PARP1) is an important first responder in the mechanism of DNA repair in eukaryotic cells. It is also a validated drug target, with four different PARP inhibitors (PARPi) approved for the treatment of BRCA-negative cancers. Despite past efforts, many aspects of PARPi are poorly understood, in particular their ability to trap PARP1 on chromatin and the relationships between their potencies, cellular toxicities, and trapping efficiencies. Because PARP trapping is widely believed to originate in allosteric coupling between DNA binding and the catalytic site, we further investigated the binding properties of PARP1 to a model for DNA with a double-strand break in the presence and absence of PARPi. Specifically, we have used sequential mixing stopped-flow spectroscopy to identify a slow conformational change that follows rapid DNA binding. Using a range of DNA concentrations and different mutants of PARP1 we demonstrate that this conformational change is one of the steps of the "monkey bar mechanism" that promotes DNA-dependent dissociation of DNA. This conformational change also corresponds to the previously identified conformational change associated with DNA-dependent activation of PARP1. Despite linking the conformational change associated with DNA binding and release to DNA activation, we find no evidence for PARPi perturbing this allosteric coupling.

Mosaic Cooperativity in Slow Polypeptide Topological Isomerization Revealed by Residue-Specific NMR Thermodynamic Analysis.

Slow polypeptide conformational changes on time scales of >1 s are generally assumed to be highly cooperative two-state transitions, reflecting the high energy barrier. However, few experimental characterizations have tested the validity of this assumption. We performed residue-specific NMR thermodynamic analysis of the 27-residue lantibiotic peptide, nukacin ISK-1, to characterize the isomerization between two topological states on the second time scale. Unexpectedly, the thermal transition behaviors were distinct among peptide regions, indicating that the topological isomerization process is a mosaic of different degrees of cooperativity. The conformational change path between the two NMR structures was deduced by a targeted molecular dynamics simulation. The unique side-chain threading motions through the monosulfide rings are the structural basis of the high energy barrier, and the nonlocal interactions in the hydrophobic core are the structural basis of the cooperativity. Taken together, we provide an energetic description of the topological isomerization of nukacin ISK-1.

Chromatographic behavior of bivalent bispecific antibodies on hydrophobic interaction chromatography columns

The elution behavior of bivalent bispecific antibodies (BiSAb) comprising an immunoglobulin G framework genetically fused to a pair of single chain variable fragments (scFvs) was studied on hydrophobic interaction chromatography (HIC) columns using ammonium sulfate gradients. Each of the BiSAb molecules studied exhibited a three-peak elution behavior regardless of the location of scFv attachment to the framework IgG. Collecting and re-injecting each of the isolated peaks and eluting with the same gradient resulted in the same three-peak profile indicating that the behavior is reversible. Analogous behavior was observed for HIC resins with different functional ligands, matrix structures, and particle sizes. Residence time, operating temperature, and hold time were shown to affect the elution behavior. While three peaks were obtained at short residence times and room temperature, residence times longer than about 27 min or operating at 45 °C resulted in a single merged peak indicating that the underlying mechanism occurs on time scales comparable to that of chromatographic separation. Holding the protein on the resins prior to elution enriched the late eluting peak indicating that multiple binding states formed on the chromatographic surface are responsible for this behavior. Tryptophan auto-fluorescence measurements show that stronger binding forms have increased solvent exposure indicating that surface-catalyzed conformational changes play a role. A model was developed to describe the interplay of chromatographic separation and slow conformational changes.

Insights on the kinetics of concanavalin A adsorption on platinum and glassy carbon electrodes from electrochemical impedance spectroscopy data

Electrochemical impedance spectroscopy was applied to investigate the adsorption of concanavalin A on glassy carbon and platinum electrodes. A redox probe, K4Fe(CN)6/K3Fe(CN)6, was used to yield a well defined charge transfer resistance across the electrode/solution interface. Adsorption on glassy carbon electrode was a diffusion controlled process during its initial stages. From the time dependence of the electrode coverage, by means of a simple rate law and the Stoke-Einstein equation, it was possible to estimate a protein effective radius of 3.7 nm and to estimate the protein monolayer coverage of 2 mg m–2. On platinum electrode the kinetics could be explained in terms of a fast initial adsorption, followed by a slow protein conformational change. The locked protein (Concanavalin A with bound Ca2+ and Mn2+) adsorbs faster than the unlocked protein. The presence of an oxide layer on the electrode seems to affect more the unlocked protein than the locked one. It is suggested that the Ca2+ and Mn2+ ions present in the locked protein may play a role on the adsorption process.

Stepwise activation of a class C GPCR begins with millisecond dimer rearrangement

G protein-coupled receptors (GPCRs) are key biological switches that transmit both internal and external stimuli into the cell interior. Among the GPCRs, the "light receptor" rhodopsin has been shown to activate with a rearrangement of the transmembrane (TM) helix bundle within ∼1 ms, while all other receptors are thought to become activated within ∼50 ms to seconds at saturating concentrations. Here, we investigate synchronous stimulation of a dimeric GPCR, the metabotropic glutamate receptor type 1 (mGluR1), by two entirely different methods: (i) UV light-triggered uncaging of glutamate in intact cells or (ii) piezo-driven solution exchange in outside-out patches. Submillisecond FRET recordings between labels at intracellular receptor sites were used to record conformational changes in the mGluR1. At millimolar ligand concentrations, the initial rearrangement between the mGluR1 subunits occurs at a speed of τ1 ∼ 1-2 ms and requires the occupancy of both binding sites in the mGluR1 dimer. These rapid changes were followed by significantly slower conformational changes in the TM domain (τ2 ∼ 20 ms). Receptor deactivation occurred with time constants of ∼40 and ∼900 ms for the inter- and intrasubunit conformational changes, respectively. Together, these data show that, at high glutamate concentrations, the initial intersubunit activation of mGluR1 proceeds with millisecond speed, that there is loose coupling between this initial step and activation of the TM domain, and that activation and deactivation follow a cyclic pathway, including-in addition to the inactive and active states-at least two metastable intermediate states.

Structure and dynamics of the platelet integrin-binding C4 domain of von Willebrand factor

AbstractVon Willebrand factor (VWF) is a key player in the regulation of hemostasis by promoting recruitment of platelets to sites of vascular injury. An array of 6 C domains forms the dimeric C-terminal VWF stem. Upon shear force activation, the stem adopts an open conformation allowing the adhesion of VWF to platelets and the vessel wall. To understand the underlying molecular mechanism and associated functional perturbations in disease-related variants, knowledge of high-resolution structures and dynamics of C domains is of paramount interest. Here, we present the solution structure of the VWF C4 domain, which binds to the platelet integrin and is therefore crucial for the VWF function. In the structure, we observed 5 intra- and inter-subdomain disulfide bridges, of which 1 is unique in the C4 domain. The structure further revealed an unusually hinged 2-subdomain arrangement. The hinge is confined to a very short segment around V2547 connecting the 2 subdomains. Together with 2 nearby inter-subdomain disulfide bridges, this hinge induces slow conformational changes and positional alternations of both subdomains with respect to each other. Furthermore, the structure demonstrates that a clinical gain-of-function VWF variant (Y2561) is more likely to have an effect on the arrangement of the C4 domain with neighboring domains rather than impairing platelet integrin binding.

Insights into the Cooperative Nature of ATP Hydrolysis in Actin Filaments

Actin filaments continually assemble and disassemble within a cell. Assembled filaments “age” as a bound nucleotide ATP within each actin subunit quickly hydrolyzes followed by a slower release of the phosphate Pi, leaving behind a bound ADP. This subtle change in nucleotide state of actin subunits affects filament rigidity as well as its interactions with binding partners. We present here a systematic multiscale ultra-coarse-graining approach that provides a computationally efficient way to simulate a long actin filament undergoing ATP hydrolysis and phosphate-release reactions while systematically taking into account available atomistic details. The slower conformational changes and their dependence on the chemical reactions are simulated with the ultra-coarse-graining model by assigning internal states to the coarse-grained sites. Each state is represented by a unique potential surface of a local heterogeneous elastic network. Internal states undergo stochastic transitions that are coupled to conformations of the underlying molecular system. The model reproduces mechanical properties of the filament and allows us to study whether conformational fluctuations in actin subunits produce cooperative filament aging. We find that the nucleotide states of neighboring subunits modulate the reaction kinetics, implying cooperativity in ATP hydrolysis and Pi release. We further systematically coarse grain the system into a Markov state model that incorporates assembly and disassembly, facilitating a direct comparison with previously published models. We find that cooperativity in ATP hydrolysis and Pi release significantly affects the filament growth dynamics only near the critical G-actin concentration, whereas far from it, both cooperative and random mechanisms show similar growth dynamics. In contrast, filament composition in terms of the bound nucleotide distribution varies significantly at all monomer concentrations studied. These results provide new insights, to our knowledge, into the cooperative nature of ATP hydrolysis and Pi release and the implications it has for actin filament properties, providing novel predictions for future experimental studies.

Classifying antimicrobial and multifunctional peptides with Bayesian network models

AbstractBayesian network models are finding success in characterizing enzyme‐catalyzed reactions, slow conformational changes, predicting enzyme inhibition, and genomics. In this work, we apply them to statistical modeling of peptides by simultaneously identifying amino acid sequence motifs and using a motif‐based model to clarify the role motifs may play in antimicrobial activity. We construct models of increasing sophistication, demonstrating how chemical knowledge of a peptide system may be embedded without requiring new derivation of model fitting equations after changing model structure. These models are used to construct classifiers with good performance (94% accuracy, Matthews correlation coefficient of 0.87) at predicting antimicrobial activity in peptides, while at the same time being built of interpretable parameters. We demonstrate use of these models to identify peptides that are potentially both antimicrobial and antifouling, and show that the background distribution of amino acids could play a greater role in activity than sequence motifs do. This provides an advancement in the type of peptide activity modeling that can be done and the ease in which models can be constructed.

Slow luminescence kinetics of semi-synthetic aequorin: expression, purification and structure determination of cf3-aequorin.

cf3-Aequorin is one of the semi-synthetic aequorins that was produced by replacing 2-peroxycoelenterazine (CTZ-OOH) in native aequorin with a 2-peroxycoelenterazine analog, and it was prepared using the C2-modified trifluoromethyl analog of coelenterazine (cf3-CTZ) and the histidine-tagged apoaequorin expressed in Escherichia coli cells. The purified cf3-aequorin showed a slow luminescence pattern with half-decay time of maximum intensities of luminescence of 5.0 s. This is much longer than that of 0.9 s for native aequorin, and its luminescence capacity was estimated to be 72.8% of that of native aequorin. The crystal structure of cf3-aequorin was determined at 2.15 Å resolution. The light source of 2-peroxytrifluoromethylcoelenterazine (cf3-CTZ-OOH) was stabilized by the hydrogen-bonding interactions at the C2-peroxy moiety and the p-hydroxy moiety at the C6-phenyl group. In native aequorin, three water molecules contribute to stabilizing CTZ-OOH through hydrogen bonds. However, cf3-aequorin only contained one water molecule, and the trifluoromethyl moiety at the C2-benzyl group of cf3-CTZ-OOH interacted with the protein by van der Waals interactions. The slow luminescence kinetics of cf3-aequorin could be explained by slow conformational changes due to the bulkiness of the trifluoromethyl group, which might hinder the smooth cleavage of hydrogen bonds at the C2-peroxy moiety after the binding of Ca2+ to cf3-aequorin.