Intermediate State Research Articles

Dimethylamine Copper(I) Halide Single Crystals: Structure, Physical Properties, and Scintillation Performance.

Hybrid copper(I) halides have garnered a significant amount of attention as potential substitutes in luminescence and scintillation applications. Herein, we report the discovery and crystal growth of new zero-dimensional compounds, (C2H8N)3Cu2I5 and (C2H8N)4Cu2Br6. The bromide and iodide have a triclinic structure with space group P1̅ and an orthorhombic structure with space group Pnma, respectively. (C2H8N)3Cu2I5 exhibits cyan emission peaking at 504 nm with a photoluminescence quantum yield (PLQY) of 34.79%, while (C2H8N)4Cu2Br6 shows yellowish-green emission peaking at 537 nm with a PLQY of 38.45%. The temperature-dependent photoluminescence data of both compounds were fitted to theoretical models, revealing that nonradiative intermediate states significantly affect thermal quenching and antiquenching. Electron-phonon interactions, the origin of emission line width broadening and peak shifting, were also investigated via fittings. The scintillation properties of (C2H8N)3Cu2I5 were evaluated, and an X-ray imaging device was successfully fabricated using (C2H8N)3Cu2I5. This work demonstrates the potentiality of copper halides in lighting and X-ray imaging applications.

How to Sample Dozens of Substitutions per Site with λ Dynamics.

Alchemical free energy methods are useful in computer-aided drug design and computational protein design because they provide rigorous statistical mechanics-based estimates of free energy differences from molecular dynamics simulations. λ dynamics is a free energy method with the ability to characterize combinatorial chemical spaces spanning thousands of related systems within a single simulation, which gives it a distinct advantage over other alchemical free energy methods that are mostly limited to pairwise comparisons. Recently developed methods have improved the scalability of λ dynamics to perturbations at many sites; however, the size of chemical space that can be explored at each individual site has previously been limited to fewer than ten substituents. As the number of substituents increases, the volume of alchemical space corresponding to nonphysical alchemical intermediates grows exponentially relative to the size corresponding to the physical states of interest. Beyond nine substituents, λ dynamics simulations become lost in an alchemical morass of intermediate states. In this work, we introduce new biasing potentials that circumvent excessive sampling of intermediate states by favoring sampling of physical end points relative to alchemical intermediates. Additionally, we present a more scalable adaptive landscape flattening algorithm for these larger alchemical spaces. Finally, we show that this potential enables more efficient sampling in both protein and drug design test systems with up to 24 substituents per site, enabling, for the first time, simultaneous simulation of all 20 amino acids.

NucMACC: An MNase-seq pipeline to identify structurally altered nucleosomes in the genome.

Micrococcal nuclease sequencing is the state-of-the-art method for determining chromatin structure and nucleosome positioning. Data analysis is complex due to the AT-dependent sequence bias of the endonuclease and the requirement for high sequencing depth. Here, we present the nucleosome-based MNase accessibility (nucMACC) pipeline unveiling the regulatory chromatin landscape by measuring nucleosome accessibility and stability. The nucMACC pipeline represents a systematic and genome-wide approach for detecting unstable ("fragile") nucleosomes. We have characterized the regulatory nucleosome landscape in Drosophila melanogaster, Saccharomyces cerevisiae, and mammals. Two functionally distinct sets of promoters were characterized, one associated with an unstable nucleosome and the other being nucleosome depleted. We show that unstable nucleosomes present intermediate states of nucleosome remodeling, preparing inducible genes for transcriptional activation in response to stimuli or stress. The presence of unstable nucleosomes correlates with RNA polymerase II proximal pausing. The nucMACC pipeline offers unparalleled precision and depth in nucleosome research and is a valuable tool for future nucleosome studies.

Structural and mechanistic analysis of Ca2+-dependent regulation of transglutaminase 2 activity using a Ca2+-bound intermediate state

Mammalian transglutaminases, a family of Ca2+-dependent proteins, are implicated in a variety of diseases. For example, celiac disease (CeD) is an autoimmune disorder whose pathogenesis requires transglutaminase 2 (TG2) to deamidate select glutamine residues in diet-derived gluten peptides. Deamidation involves the formation of transient γ-glutamyl thioester intermediates. Recent studies have revealed that in addition to the deamidated gluten peptides themselves, their corresponding thioester intermediates are also pathogenically relevant. A mechanistic understanding of this relevance is hindered by the absence of any structure of Ca2+-bound TG2. We report the X-ray crystallographic structure of human TG2 bound to an inhibitory gluten peptidomimetic and two Ca2+ ions in sites previously designated as S1 and S3. Together with additional structure-guided experiments, this structure provides a mechanistic explanation for how S1 regulates formation of an inhibitory disulfide bond in TG2, while also establishing that S3 is essential for γ-glutamyl thioester formation. Furthermore, our crystallographic findings and associated analyses have revealed that i) two interacting residues, H305 and E363, play a critical role in resolving the thioester intermediate into an isopeptide bond (transamidation) but not in thioester hydrolysis (deamidation); and ii) residues N333 and K176 stabilize preferred TG2 substrates and inhibitors via hydrogen bonding to nonreactive backbone atoms. Overall, the intermediate-state conformer of TG2 reported here represents a superior model to previously characterized conformers for both transition states of the TG2-catalyzed reaction.

Unconventional hcp/fcc Nickel Heteronanocrystal with Asymmetric Convex Sites Boosts Hydrogen Oxidation

Developing non‐platinum group metal catalysts for the sluggish hydrogen oxidation reaction (HOR) is critical for alkaline fuel cells. To date, Ni‐based materials are the most promising candidates but still suffer from insufficient performance. Herein, we report an unconventional hcp/fcc Ni (u‐hcp/fcc Ni) heteronanocrystal with multiple epitaxial hcp/fcc heterointerfaces and coherent twin boundaries, generating rugged surfaces with plenty of asymmetric convex sites. Systematic analyses discover that such convex sites enable the adsorption of *H in unusual bridge positions with weakened binding energy, circumventing the over‐strong *H adsorption on traditional hollow positions, and simultaneously stabilizing interfacial *H2O. It thus synergistically optimizes the HOR thermodynamic process as well as reduces the kinetic barrier of the rate‐determining Volmer step. Consequently, the developed u‐hcp/fcc Ni exhibits the top‐rank alkaline HOR activity with a mass activity of 40.6 mA mgNi‐1 (6.3 times higher than fcc Ni control) together with superior stability and high CO‐tolerance. These results provide a paradigm for designing high‐performance catalysts by shifting the adsorption state of intermediates through configuring surface sites.

Insights into the oxidative thermal stability of mesoporous triazine‐based organic polymers: Kinetics and thermodynamic parameters

AbstractThis study investigates the thermal degradation kinetics of mesoporous triazine‐based polymers, namely triazine‐amine and triazine‐ether polymers. The synthesis, physicochemical characterization, and catalytic applications of these polymers were discussed in our previous report. Herein, the thermal stability parameters, including kinetic triplets and thermodynamic parameters, were determined using thermogravimetric analysis (TGA) and non‐isothermal mathematical approximations such as Coats‐Redfern, Broido, and Horowitz–Metzger methods. Triazine‐ether polymers exhibit thermal stability within the range of 200°C–300°C, while triazine‐amine polymer demonstrates superior thermal stability, reaching up to 450°C. According to the Coats‐Redfern method, the degradation follows reaction orders of 0.5 ≤ n ≤ 1. The activation energy of triazine‐amine polymer is notably high, particularly at the third degradation stage (e.g., 89.0 kJ/mol by the Broido method), attributed to its high nitrogen content. Conversely, the higher carbon content of triazine‐ether polymers reduces their activation energy to approximately 30 kJ/mol at all stages and thus, facilitates the degradation process. Thermodynamically, the degradation process is favorable yet non‐spontaneous, with intermediate states of the polymers exhibiting higher entropy, indicative of their enhanced degradation capability.

Long term variability of Cygnus X-1

The Neutron Star Interior Composition Explorer (NICER) monitoring campaign of Cyg X-1 allows us to study its spectral-timing behavior at energies < 1 keV across all states. The hard state power spectrum can be decomposed into two main broad Lorentzians with a transition at around 1 Hz. The lower-frequency Lorentzian is the dominant component at low energies. The higher-frequency Lorentzian begins to contribute significantly to the variability above 1.5 keV and dominates at high energies. We show that the low- and high-frequency Lorentzians likely represent individual physical processes. The lower-frequency Lorentzian can be associated with a (possibly Comptonized) disk component, while the higher-frequency Lorentzian is clearly associated with the Comptonizing plasma. At the transition of these components, we discover a low-energy timing phenomenon characterized by an abrupt lag change of hard (≳2 keV) with respect to soft (≲1.5 keV) photons, accompanied by a drop in coherence, and a reduction in amplitude of the second broad Lorentzian. The frequency of the phenomenon increases with the frequencies of the Lorentzians as the source softens and cannot be seen when the power spectrum is single-humped. A comparison to transient low-mass X-ray binaries shows that this feature does not only appear in Cyg X-1, but that it is a general property of accreting black hole binaries. In Cyg X-1, we find that the variability at low and high energies is overall highly coherent in the hard and intermediate states. The high coherence shows that there is a process at work which links the variability, suggesting a physical connection between the accretion disk and Comptonizing plasma. This process fundamentally changes in the soft state, where strong red noise at high energies is incoherent to the variability at low energies.

Stabilization of Non-Native Folds and Programmable Protein Gelation in Compositionally Designed Deep Eutectic Solvents.

Proteins are adjustable units from which biomaterials with designed properties can be developed. However, non-native folded states with controlled topologies are hardly accessible in aqueous environments, limiting their prospects as building blocks. Here, we demonstrate the ability of a series of anhydrous deep eutectic solvents (DESs) to precisely control the conformational landscape of proteins. We reveal that systematic variations in the chemical composition of binary and ternary DESs dictate the stabilization of a wide range of conformations, that is, compact globular folds, intermediate folding states, or unfolded chains, as well as controlling their collective behavior. Besides, different conformational states can be visited by simply adjusting the composition of ternary DESs, allowing for the refolding of unfolded states and vice versa. Notably, we show that these intermediates can trigger the formation of supramolecular gels, also known as eutectogels, where their mechanical properties correlate to the folding state of the protein. Given the inherent vulnerability of proteins outside the native fold in aqueous environments, our findings highlight DESs as tailorable solvents capable of stabilizing various non-native conformations on demand through solvent design.

Single-cell sequencing unveils the impact of aging on the progenitor cell diversity in the telencephalon of the female killifish N. furzeri.

The African turquoise killifish (Nothobranchius furzeri) combines a short lifespan with spontaneous age-associated loss of neuro-regenerative capacity, an intriguing trait atypical for a teleost. The impact of aging on the cellular composition of the adult stem cell niches, leading to this dramatic decline in the postnatal neuro- and gliogenesis, remains elusive. Single-cell RNA sequencing of the telencephalon of young adult female killifish of the short-lived GRZ-AD strain unveiled progenitors of glial and non-glial nature, different excitatory and inhibitory neuron subtypes, as well as non-neural cell types. Sub-clustering of the progenitors identified four radial glia (RG) cell types, two non-glial progenitor (NGP) and four intermediate (intercell) cell states. Two astroglia-like, one ependymal, and one neuroepithelial-like (NE) RG subtype were found at different locations in the forebrain in line with their role, while proliferative, active NGPs were spread throughout. Lineage inference pointed to NE-RG and NGPs as start and intercessor populations for glio- and neurogenesis. Upon aging, single-cell RNA sequencing revealed major perturbations in the proportions of the astroglia and intercell states, and in the molecular signatures of specific subtypes, including altered MAPK, mTOR, Notch, and Wnt pathways. This cell catalog of the young regeneration-competent killifish telencephalon, combined with the evidence for aging-related transcriptomic changes, presents a useful resourceto understand the molecular basis of age-dependent neuroplasticity. This data is also available through an online database (killifishbrain_scseq).

Electrocatalytic Reduction of Nitrate to Ammonia at Oxidized Vanadium Surfaces with V(3+) and V(4+) Oxidation States

The electrochemical reduction of nitrate to ammonia is of interest as an energy/environmentally friendly source of ammonia for agriculture and energy applications and as a route toward groundwater purification. We report in situ photoemission data, electrochemical results, and density functional theory calculations that demonstrate vanadium oxide—prepared by ambient exposure of V metal, with a distribution of surface V3+ and V4+ oxidation states—specifically adsorbs and reduces nitrate to ammonia at pH 3.2 at cathodic potentials. Negligible cathodic activity in the absence of NO3 − indicates high selectivity with respect to non-nitrate reduction processes. In situ photoemission data indicate that nitrate adsorption and reduction to adsorbed NO2 is a key step in the reduction process. NO3RR activity is also observed at pH 7, albeit at a much slower rate. The results indicate that intermediate (non-d0) oxidation states are important for both molecular nitrogen and nitrate reduction to ammonia.

Polarization at the compositional interface in Nb-doped metastable TiO2-SnO2 solid solutions

Polarization architecture was incorporated into metastable Nb-doped TiO2-SnO2 to deliver electron accumulation at the localized TiO2-SnO2 compositionally fluctuating interface. Specimens were quenched from various holding temperatures to ambient temperatures in air to avoid bimodal decomposition into TiO2 and SnO2 endmembers. At the lowest sintering temperature of 1,400 °C, the mixed phase containing TiO2- and SnO2-rich compositions existed as an intermediate state to the single-phase solid solution. The phase boundary became more ambiguous with increasing sintering temperatures, and the compositional fluctuation size reduced to single nanometers at 1,500 °C. The permittivity due to the interfacial polarization, ε interface, increased steadily with increasing sintering temperature. The larger ε interface values at higher temperatures are attributed to the greater density of the compositionally fluctuating phase interface, which leads to greater electron accumulation at the energy barrier between the two semiconducting layers.

Single Atom Ag Bonding Between PF3T Nanocluster and TiO2 Leads the Ultra-Stable Visible-Light-Driven Photocatalytic H2 Production.

Atomic Ag cluster bonding is employed to reinforce the interface between PF3T nano-cluster and TiO2 nanoparticle. With an optimized Ag loading (Ag/TiO2=0.5wt%), the Ag atoms will uniformly disperse on TiO2 thus generating a high density of intermediate states in the band gap to form the electron channel between the terthiophene group of PF3T and the TiO2 in the hybrid composite (denoted as T@Ag05-P). The former expands the photon absorption band width and the latter facilitates the core-hole splitting by injecting the photon excited electron (from the excitons in PF3T) into the conduction band (CB) of TiO2. These characteristics enable the high efficiency of H2 production to 16580µmolh-1g-1 and photocatalysis stability without degradation under visible light exposure for 96h. Compared to that of hybrid material without Ag bonding (TiO2@PF3T), the H2 production yield and stability are improved by 4.1 and 18.2-fold which shows the best performance among existing materials in similar component combination and interfacial reinforcement. The unique bonding method offers a new prospect to accelerate the development of photocatalytic hydrogen production technologies.

Local strain heterogeneity associated with Al/Si ordering in anorthite, CaAl2Si2O8, with implications for thermodynamic mixing behavior and trace element partitioning in plagioclase feldspars

Abstract Hard Mode IR powder absorption spectroscopy has been used to characterize local strain relaxation associated with Al/Si ordering in a suite of synthetic anorthite samples with structural states that vary from a high degree of Al/Si order through a metastable incommensurate structure at intermediate states of order to long-range order with I1 symmetry. The dominant feature accompanying the changing structural states is line broadening, which has been quantified by autocorrelation analysis and is attributed to local heterogeneous strain variations on a length scale of at least 1–5 unit cells. The autocorrelation results are consistent with contributions to the line broadening as being due to order parameters for both the C1 → I1 and I1 → P1 transitions, which couple biquadratically, λQod2Qdispl2. Close correlation with enthalpy variations from previously published calorimetric data indicates that the driving force for ordering can be understood in terms of elimination of strain fields arising from accommodating more or less rigid AlO4 and SiO4 tetrahedra in the feldspar framework. The metastable incommensurate structure of anorthite is closely analogous to the stable incommensurate structure that develops at intermediate compositions in the plagioclase solid solution, confirming that the same strain relaxation mechanism dominates the properties and behavior of all structural states across the solid solution. Elimination of strain heterogeneity by ordering on the basis of I1 symmetry determines the form of non-ideal mixing shown by the solid solution at high temperatures, and changes in elastic properties may contribute to a break in the slope of partitioning of trace elements between crystals and melt.

예수 부활과 초대교회의 해석

This paper examines the issue of the historicity of Jesus' resurrection and discusses the early church's interpretation of the resurrection. The core controversy surrounding the historicity of Jesus' resurrection is whether Jesus was truly resurrected in the flesh. While in Galilee the disciples experienced an apparition of Jesus, in Jerusalem women discovered an empty tomb and experienced an apparition. When the disciples returned to Jerusalem and heard about the empty tomb, the apparitions and the empty tomb complemented each other and solidified the belief in resurrection. However, as Paul said (1 Cor 15,16), without the Jewish faith in the resurrection, Jesus would not have been resurrected. Since Jesus himself shared the Jewish faith in the resurrection, his death did not end in vain and he could predict God's victory. The issue of bodily resurrection raises questions about the steps prior to resurrection. Jesus taught that people live in spirit form even after death (Q 12,4-5). Jesus taught that humans are always before God, whether alive in body or alive in spirit after death (cf. Mk 12,27). The Jesus' proclamation of the Kingdom of God being both future and present is based on his conviction that everything is alive before God. Therefore everything is already in a continuous process toward the eschatological goal. The intermediate state between individual death and resurrection is seen as a continuous process toward eschatological resurrection. Jesus and the early Christians believed in an intermediate state of survival of the spirit after death. The reason the early church was able to solve the problem of delaying the Second Coming (Mk 13,32; 2Peter 3,4) was also due to the belief in the intermediate stage of being with Christ even after individual death (cf. Lk 23:43; Phil 1:23). The early Christians understood the period before the Second Coming as a time for the church to prepare for eschatological restoration (Acts 3:20-21) and actively carried out missionary activities to participate in the process of salvation history (Acts 1:8). Luke gives a narrative of birth of the church through the resurrection, ascension, and sending of the Holy Spirit. Mark and Matthew, who do not mention the ascension, emphasize that the crucified Jesus was resurrected and is still with his disciples. The early Christians who have experienced the resurrection and apparitions of Jesus as a force for leading history proclaimed the gospel of resurrection. Therefore, they were able to further commit themselves to the historical process toward the new creation for the final restoration of the whole world.

A neutralizing antibody prevents postfusion transition of measles virus fusion protein.

Measles virus (MeV) presents a public health threat that is escalating as vaccine coverage in the general population declines and as populations of immunocompromised individuals, who cannot be vaccinated, increase. There are no approved therapeutics for MeV. Neutralizing antibodies targeting viral fusion are one potential therapeutic approach but have not yet been structurally characterized or advanced to clinical use. We present cryo-electron microscopy (cryo-EM) structures of prefusion F alone [2.1-angstrom (Å) resolution], F complexed with a fusion-inhibitory peptide (2.3-Å resolution), F complexed with the neutralizing and protective monoclonal antibody (mAb) 77 (2.6-Å resolution), and an additional structure of postfusion F (2.7-Å resolution). In vitro assays and examination of additional EM classes show that mAb 77 binds prefusion F, arrests F in an intermediate state, and prevents transition to the postfusion conformation. These structures shed light on antibody-mediated neutralization that involves arrest of fusion proteins in an intermediate state.

Fe7S8 coupled with VS4 heterogeneous interface engineering driven by FeV bimetallic MOFs: An efficient all-pH and durable hydrogen evolution

Rational design and preparation of a multiphase electrocatalyst for hydrogen evolution reaction (HER) has become a hot research topic, while applicable and pH versatility of vanadium tetrasulfide (VS4) and heptairon octasulfide (Fe7S8) composites have rarely been reported. Here, the facile topological sulfide self-template sacrifice method using FeV bimetallic MOFs is designed to obtain Fe7S8 coupled with VS4 heterostructures, enhancing the electron precipitation in the catalysts and attracts electrons to migrate. According to DFT simulations, the electronic coupling at the atomic orbital level and the modulation of interfacial electrons among various interfaces play a crucial role in enhancing the intermediate state process of the hydrogen evolution reaction (HER) across the entire pH range, promoting the optimal d-band centroid value (εd). Reassuringly, the prepared 3D Fe7S8/VS4 electrodes possessed excellent performances of η10 = 53 mV, η10 = 135 mV and η10 = 38 mV in a conventional three-electrode configuration in a 1 M KOH, 1 M Na2SO4, and 0.5 M H2SO4, and the stabilized currents can all be maintained for 48 h. This innovative design of in situ heterostructured materials constructed from dual transition metal sulfides provides inspiring ideas for the preparation of all-pH catalysts.

Brain Network Dynamics in Women With Primary Dysmenorrhea During the Pain-Free Periovulation Phase

The human brain is a dynamic system that shows frequency-specific features. Neuroimaging studies have shown that both healthy individuals and those with chronic pain disorders experience pain influenced by various processes that fluctuate over time. Primary dysmenorrhea (PDM) is a chronic visceral pain that disrupts the coordinated activity of brain’s functional network. However, it remains unclear whether the dynamic interactions across the whole-brain network over time and their associations with neurobehavioral symptoms are dependent on the frequency bands in patients with PDM during the pain-free periovulation phase. In this study, we used an energy landscape analysis to examine the interactions over time across the large-scale network in a sample of 59 patients with PDM and 57 healthy controls (HCs) at different frequency bands. Compared with HCs, patients with PDM exhibit aberrant brain dynamics, with more significant differences in the slow-4 frequency band. Patients with PDM show more indirect neural transition counts due to an unstable intermediate state, whereas neurotypical brain activity frequently transitions between 2 major states. This data-driven approach further revealed that the brains of individuals with PDM have more abnormal brain dynamics than HCs. Our results suggested that unstable brain dynamics were associated with the strength of brain functional segregation and the Pain Catastrophizing Scale score. Our findings provide preliminary evidence that atypical dynamics in the functional network may serve as a potential key feature and biological marker of patients with PDM during the pain-free phase. PerspectiveWe applied energy landscape analysis on brain-imaging data to identify relatively stable and dominant brain activity patterns for patients with PDM. More atypical brain dynamics were found in the slow-4 band and were related to the strength of functional segregation, providing new insights into the dysfunction brain dynamics.

Hydrogen bond symmetrisation in D2O ice observed by neutron diffraction

Hydrogen bond symmetrisation is the phenomenon where a hydrogen atom is located at the centre of a hydrogen bond. Theoretical studies predict that hydrogen bonds in ice VII eventually undergo symmetrisation upon increasing pressure, involving nuclear quantum effect with significant isotope effect and drastic changes in the elastic properties through several intermediate states with varying hydrogen distribution. Despite numerous experimental studies conducted, the location of hydrogen and hence the transition pressures reported up to date remain inconsistent. Here we report the atomic distribution of deuterium in D2O ice using neutron diffraction above 100 GPa and observe the transition from a bimodal to a unimodal distribution of deuterium at around 80 GPa. At the transition pressure, a significant narrowing of the peak widths of 110 is also observed, attributed to the structural relaxation by the change of elastic properties.

Competition and perspectives of protein-carbon and specific inorganic ecosystems on Earth

The article presents in a parametric comparison the state and development of two ecosystems, one of which is based on carbonaceous life forms, and the second – on metalloid and metallic elements available on the planet and at the disposal of man. It is possible to come to a hypothetical assumption about possible competition between these systems within the selected parameters. Well-known studies unbiasedly confirm the possibilities for such competition. Our research is based on the ergatic «human-machine» system as one that contains two main elements of the ecosystems under study. The proposed parametric model, reflecting the main mutual competitive functions of man and «machine» – reduced power, conditional indicators of intelligence in the chronological aspect, made it possible to assess both their real and potential capabilities and draw conclusions about the prospects of these two conditional ecosystems. One of the conclusions of such studies was a non-trivial thesis about the possible intermediate state of man in the long-term evolutionary development of the mind on the planet. and that he is beginning to lose competition with the specific forms of the inorganic world he has created. The arguments are comparative data: life expectancy, the ability to irrational self-elimination, methods of reproduction, the ability to develop the mind and memory, the causes of environmental degradation and its impact on humans, changes in the motivation of human life, the programmed rejection of the natural protein-carbon environment in favor of an unnatural specialized inorganic world. Logic dictates that perhaps man is not the most ideal evolutionary creation in nature, because it is already becoming clear that in addition to carbon, but with the help of man, other forms of intelligence and consciousness are possible even on Earth itself. It is shown that specific forms of inorganic matter, with a vector of development towards artificial intelligence, are protected from the main disadvantage of humans – a short life cycle and the ability to self-destruct. The modern evolutionary vector is not its last stage, it is aimed at the development of specific forms of the inorganic world, which at a certain time will be capable of activity in parallel with man, but no less effective

The Structural Stability of Membrane Proteins Revisited: Combined Thermodynamic and Spectral Phasor Analysis of SDS-induced Denaturation of a Thermophilic Cu(I)-transport ATPase

Assessing membrane protein stability is among the major challenges in protein science due to their inherent complexity, which complicates the application of conventional biophysical tools. In this work, sodium dodecyl sulfate-induced denaturation of AfCopA, a Cu(I)-transport ATPase from Archaeoglobus fulgidus, was explored using a combined model-free spectral phasor analysis and a model-dependent thermodynamic analysis. Decrease in tryptophan and 1-anilino-naphthalene-8-sulfonate fluorescence intensity, displacements in the spectral phasor space, and the loss of ATPase activity were reversibly induced by this detergent. Refolding from the SDS-induced denatured state yields an active enzyme that is functionally and spectroscopically indistinguishable from the native state of the protein. Phasor analysis of Trp spectra allowed us to identify two intermediate states in the SDS-induced denaturation of AfCopA, a result further supported by principal component analysis. In contrast, traditional thermodynamic analysis detected only one intermediate state, and including the second one led to overparameterization. Additionally, ANS fluorescence spectral analysis detected one more intermediate and a gradual change at the level of the hydrophobic transmembrane surface of the protein. Based on this evidence, a model for acquiring the native structure of AfCopA in a membrane-like environment is proposed.