A bifunctional primitive strategy induces enhancements of large second harmonic generation and wide UV transmittance in rare-earth borates containing [B5O10] groups.

Strong second-harmonic generation (SHG) and a short ultraviolet (UV) cutoff edge are two crucial yet often conflicting parameters that must be finely tuned in the exploration of nonlinear optical (NLO) materials. In this study, two new rare earth borate NLO crystals, K7BaSc2B15O30 (KBSBO) and Rb21Sr3.8Sc5.2B45O90 (RSSBO), were rationally designed through a bifunctional primitive strategy to achieve an optimized balance between favorable SHG efficiency and UV transparency. As anticipated, both KBSBO and RSSBO exhibit a wide UV transparency window below 190 nm. Notably, these tailored crystals display strong SHG responses, with RSSBO achieving a remarkable enhancement in SHG efficiency (2 × KDP), surpassing that of most deep-UV rare earth borates containing [B5O10] groups known to date. Theoretical calculations and structural analyses reveal that the impressive SHG activities primarily stem from the [B5O10] groups and [ScO6] polyhedra. These findings suggest promising potential for KBSBO and RSSBO crystals as beryllium-free deep UV NLO materials.

Studying protein stability in crowded environments by NMR.

Most proteins perform their functions in crowded and complex cellular environments where weak interactions are ubiquitous between biomolecules. These complex environments can modulate the protein folding energy landscape and hence affect protein stability. NMR is a nondestructive and effective method to quantify the kinetics and equilibrium thermodynamic stability of proteins at an atomic level within crowded environments and living cells. Here, we review NMR methods that can be used to measure protein stability, as well as findings of studies on protein stability in crowded environments mimicked by polymer and protein crowders and in living cells. The important effects of chemical interactions on protein stability are highlighted and compared to spatial excluded volume effects.

Progress, Challenges and Opportunities of NMR and XL-MS for Cellular Structural Biology.

The validity of protein structures and interactions, whether determined under ideal laboratory conditions or predicted by AI tools such as Alphafold2, to precisely reflect those found in living cells remains to be examined. Moreover, understanding the changes in protein structures and interactions in response to stimuli within living cells, under both normal and disease conditions, is key to grasping proteins' functionality and cellular processes. Nevertheless, achieving high-resolution identification of these protein structures and interactions within living cells presents a technical challenge. In this Perspective, we summarize the recent advancements in in-cell nuclear magnetic resonance (NMR) and in vivo cross-linking mass spectrometry (XL-MS) for studying protein structures and interactions within a cellular context. Additionally, we discuss the challenges, opportunities, and potential benefits of integrating in-cell NMR and in vivo XL-MS in future research to offer an exhaustive approach to studying proteins in their natural habitat.

Li13YGe4O16: A Mid-infrared Rare-Earth Germanate Nonlinear Optical Crystal Featuring a Broad Transmission Range and an Enlarged Band Gap.

Germanate is garnering increasing attention in the field of optoelectronics owing to its competitive optical transparency and robust stability. Herein, a novel lithium-rich rare-earth germanate, Li13YGe4O16, was fabricated for the first time using a high-temperature solution approach. This compound adopts the asymmetric space group Cmc21 (no. 36), characterized by isolated [YO6] and [GeO4] structural motifs with Li+ cations located in the channel. Notably, Li13YGe4O16 presents a short ultraviolet cutoff edge at 240 nm, indicative of an enlarged band gap of 4.96 eV and showcases a wide mid-infrared transmission region exceeding 6.0 µm. Moreover, Li13YGe4O16 features exceptional thermal stability and moderate second harmonic generation (SHG) intensity. Additionally, a theoretical analysis suggests that the distorted [YO6] octahedra. [GeO4] and [LiO4] tetrahedra play a significant role in the optical activities of Li13YGe4O16. These attributes endow Li13YGe4O16 with the potential to serve as a new mid-IR nonlinear optical (NLO) crystal and enrich the structural chemistry of germanates.

Real-Time Observation of Conformational Changes and Translocation of Endogenous Cytochrome c within Intact Mitochondria.

Cytochrome c (cyt c) is a multifunctional protein with varying conformations. However, the conformation of cyt c in its native environment, mitochondria, is still unclear. Here, we applied NMR spectroscopy to investigate the conformation and location of endogenous cyt c within intact mitochondria at natural isotopic abundance, mainly using widespread methyl groups as probes. By monitoring time-dependent chemical shift perturbations, we observed that most cyt c is located in the inner mitochondrial membrane and partially unfolded, which is distinct from its native conformation in solution. When suffering oxidative stress, cyt c underwent oxidative modifications due to increasing reactive oxygen species (ROS), weakening electrostatic interactions with the membrane, and gradually translocating into the inner membrane spaces of mitochondria. Meanwhile, the lethality of oxidatively modified cyt c to cells was reduced compared with normal cyt c. Our findings significantly improve the understanding of the molecular mechanisms underlying the regulation of ROS by cyt c in mitochondria. Moreover, it highlights the potential of NMR to monitor high-concentration molecules at a natural isotopic abundance within intact cells or organelles.

19F Nuclear Magnetic Resonance Fingerprinting Technique for Identifying and Quantifying G-Quadruplex Topology in Human Telomeric Overhangs.

G-quadruplexes (G4s) are noncanonical nucleic acid secondary structures with diverse topological features and biological roles. Human telomeric (Htelo) overhangs consisting of TTAGGG repeats can fold into G4s that adopt different topologies under physiological conditions. These G4s are potential targets for anticancer drugs. Despite intensive research, the existence and topology of G4s at Htelo overhangs in vivo are still unclear because there is no method to distinguish and quantify the topology of Htelo overhangs with native lengths that can form more than three tandem G4s in living cells. Herein, we present a novel 19F chemical shift fingerprinting technique to identify and quantify the topology of the Htelo overhangs up to five G-quadruplexes (G4s) and 120 nucleotides long both in vitro and in living cells. Our results show that longer overhang sequences tend to form stable G4s at the 5'- and 3'-ends, while the interior G4s are dynamic and "sliding" along the sequence, with TTA or 1-3 TTAGGG repeats as a linker. Each G4 in the longer overhang is conformationally heterogeneous, but the predominant ones are hybrid-2, two- or three-tetrad antiparallel, and hybrid-1 at the 5'-terminal, interior, and 3'-terminal, respectively. Additionally, we observed a distinct behavior of different lengths of telomeric sequences in living cells, suggesting that the overhang length and protein accessibility are related to its function. This technique provides a powerful tool for quickly identifying the folding topology and relative population of long Htelo overhangs, which may provide valuable insights into telomere functionality and be beneficial for structure-based anticancer drug development targeting G4s.

An ATP "Synthase" Derived from a Single Structural Domain of Bacterial Histidine Kinase.

ATP (adenosine triphosphate) is a vital energy source for living organisms, and its biosynthesis and precise concentration regulation often depend on macromolecular machinery composed of protein complexes or complicated multidomain proteins. We have identified a single-domain protein HK853CA derived from bacterial histidine kinases (HK) that can catalyze ATP synthesis efficiently. Here, we explored the reaction mechanism and multiple factors that influence this catalysis through a combination of experimental techniques and molecular simulations. Moreover, we optimized its enzymatic activity and applied it as an ATP replenishment machinery to other ATP-dependent systems. Our results broaden the understanding of ATP biosynthesis and show that the single CA domain can be applied as a new biomolecular catalyst used for ATP supply.

An efficient method for detecting membrane protein oligomerization and complex using 05SAR-PAGE.

Oligomerization is an important feature of proteins, which gives a defined quaternary structure to complete the biological functions. Although frequently observed in membrane proteins, characterizing the oligomerization state remains complicated and time-consuming. In this study, 0.05% (w/v) sarkosyl-polyacrylamide gel electrophoresis (05SAR-PAGE) was used to identify the oligomer states of the membrane proteins CpxA, EnvZ, and Ma-Mscl with high sensitivity. Furthermore, two-dimensional electrophoresis (05SAR/sodium dodecyl sulfate-PAGE) combined with western blotting and liquid chromatography-tandem mass spectrometry was successfully applied to study the complex of CpxA/OmpA in cell lysate. The results indicated that 05SAR-PAGE is an efficient, economical, and practical gel method that can be widely used for the identification of membrane protein oligomerization and the analysis of weak protein interactions.

NMR spectroscopy for metabolomics in the living system: recent progress and future challenges.

Metabolism is a fundamental process that underlies human health and diseases. Nuclear magnetic resonance (NMR) techniques offer a powerful approach to identify metabolic processes and track the flux of metabolites at the molecular level in living systems. An in vitro study through in-cell NMR tracks metabolites in real time and investigates protein structures and dynamics in a state close to their most natural environment. This technique characterizes metabolites and proteins involved in metabolic pathways in prokaryotic and eukaryotic cells. In vivo magnetic resonance spectroscopy (MRS) enables whole-organism metabolic monitoring by visualizing the spatial distribution of metabolites and targeted proteins. One limitation of these NMR techniques is the sensitivity, for which a possible improved approach is through isotopic enrichment or hyperpolarization methods, including dynamic nuclear polarization (DNP) and parahydrogen-induced polarization (PHIP). DNP involves the transfer of high polarization from electronic spins of radicals to surrounding nuclear spins for signal enhancements, allowing the detection of low-abundance metabolites and real-time monitoring of metabolic activities. PHIP enables the transfer of nuclear spin polarization from parahydrogen to other nuclei for signal enhancements, particularly in proton NMR, and has been applied in studies of enzymatic reactions and cell signaling. This review provides an overview of in-cell NMR, in vivo MRS, and hyperpolarization techniques, highlighting their applications in metabolic studies and discussing challenges and future perspectives.

Rb8Nb10Ge6O41: a new niobium-germanate crystal featuring unique one-dimensional [Nb7O30]∞ chains and wide mid-IR transparency.

Germanate oxides have garnered considerable interest owing to their diverse structural configuration and intriguing properties. Herein, we present a novel niobium germanate crystal, Rb8Nb10Ge6O41, extracted through the process of spontaneous crystallization. It showcases a unique three-dimensional (3D) structural framework composed of one-dimensional (1D) twisted [Nb7O30]∞ chains and isolated [Ge3O9] rings, arising from the divergent polymerized manifestations of [NbO6] and [GeO4] basic building blocks, respectively, marking the first instance of such a topography in germanate materials. Notably, the title compound exhibits exceptional thermal stability up to 1250 °C with a good congruent melting nature. Moreover, it achieves a short ultraviolet edge at 306 nm and a favorable infrared edge cutoff exceeding 6.2 µm, thus indicating a wide transparency window. Additionally, this study elucidates the microscopic birefringence of Rb8Nb10Ge6O41 and clarifies the intricate relationship between its structure and properties. Our findings suggest that the polymerization of distinct structural motifs within a single compound is an effective strategy for exploring novel inorganic materials.

KNa2Lu(BO3)2: A Rare-Earth Borate Crystal Characterized by an Enhanced Birefringence and Wide Ultraviolet Transparency Range.

Borate materials are of significant interest due to their versatile structural configuration and competitive ultraviolet (UV) transparency range. In this study, we present a novel rare-earth borate crystal, KNa2Lu(BO3)2, synthesized for the first time through a facile spontaneous crystallization method. It adopts the centrosymmetric space group Pnma (no. 62) and yields a unique three-dimensional (3D) structural network formed by isolated [BO3] plane triangles and distorted [LuO7] polyhedra. This compound displays excellent thermal stability up to ∼990 °C, demonstrating a favorable congruent melting nature. Moreover, KNa2Lu(BO3)2 achieves a notably short UV absorption cutoff at approximately 204 nm, yielding a large band gap of 5.58 eV. Remarkably, it showcases an enlarged birefringence of 0.044 at 1064 nm, implying its potential as a birefringent material. Moreover, density functional theory calculations demonstrate that the optical characteristics are predominantly influenced by fundamental building blocks [BO3] triangles and distorted [LuO7] polyhedra. Our findings demonstrate the potential of KNa2Lu(BO3)2 in the development of a birefringent candidate and enrich the structural chemistry of rare-earth-based borates.

In-Cell 19F†NMR of Proteins: Recent Progress and Future Opportunities.

In vitro, 19F NMR methodology is preferably selected as a complementary and straightforward method for unveiling the conformations, dynamics, and interactions of biological molecules. Its effectiveness in vivo has seen continuous improvement, addressing challenges faced by conventional heteronuclear NMR experiments on structured proteins, such as severe line broadening, low signal-to-noise ratio, and background signals. Herein, we summarize the distinctive advantages of 19F NMR, along with recent progress in sample preparation and applications within the realm of in-cell NMR. Additionally, we offer insights into the future directions and prospects of this methodology based on our understanding.

A TeZla micromixer for interrogating the early and broad folding landscape of G-quadruplex via multistage velocity descending.

Biomacromolecular folding kinetics involves fast folding events and broad timescales. Current techniques face limitations in either the required time resolution or the observation window. In this study, we developed the TeZla micromixer, integrating Tesla and Zigzag microstructures with a multistage velocity descending strategy. TeZla achieves a significant short mixing dead time (40 µs) and a wide time window covering four orders of magnitude (up to 300 ms). Using this unique micromixer, we explored the folding landscape of c-Myc G4 and its noncanonical-G4 derivatives with different loop lengths or G-vacancy sites. Our findings revealed that c-Myc can bypass folding intermediates and directly adopt a G4 structure in the cation-deficient buffer. Moreover, we found that the loop length and specific G-vacancy site could affect the folding pathway and significantly slow down the folding rates. These results were also cross-validated with real-time NMR and circular dichroism. In conclusion, TeZla represents a versatile tool for studying biomolecular folding kinetics, and our findings may ultimately contribute to the design of drugs targeting G4 structures.

Short and long range 2D 15N-15N NMR correlations among peptide groups by novel solid state dipolar mixing schemes.

A recently developed homonuclear dipolar recoupling scheme, Adiabatic Linearly FREquency Swept reCOupling (AL FRESCO), was applied to record two-dimensional (2D) 15N-15N correlations on uniformly 15N-labeled GB1 powders. A major feature exploited in these 15N-15N correlations was AL FRESCO's remarkably low RF power demands, which enabled seconds-long mixing schemes when establishing direct correlations. These 15N-15N mixing schemes proved efficient regardless of the magic-angle spinning (MAS) rate and, being nearly free from dipolar truncation effects, they enabled the detection of long-range, weak dipolar couplings, even in the presence of strong short-range dipolar couplings. This led to a connectivity information that was significantly better than that obtained with spontaneously proton-driven, 15N spin-diffusion experiments. An indirect approach producing long-range 15N-15N correlations was also tested, relying on short (ms-long) 1HN-1HN mixings schemes while applying AL FRESCO chirped pulses along the 15N channel. These indirect mixing schemes produced numerous long-distance Ni-Ni±n (n = 2 - 5) correlations, that might be useful for characterizing three-dimensional arrangements in proteins. Once again, these AL FRESCO mediated experiments proved more informative than variants based on spin-diffusion-based 1HN-1HN counterparts.

Distinct activation mechanisms of ß-arrestin-1 revealed by 19F NMR spectroscopy.

ß-Arrestins (ßarrs) are functionally versatile proteins that play critical roles in the G-protein-coupled receptor (GPCR) signaling pathways. While it is well established that the phosphorylated receptor tail plays a central role in ßarr activation, emerging evidence highlights the contribution from membrane lipids. However, detailed molecular mechanisms of ßarr activation by different binding partners remain elusive. In this work, we present a comprehensive study of the structural changes in critical regions of ßarr1 during activation using 19F NMR spectroscopy. We show that phosphopeptides derived from different classes of GPCRs display different ßarr1 activation abilities, whereas binding of the membrane phosphoinositide PIP2 stabilizes a distinct partially activated conformational state. Our results further unveil a sparsely-populated activation intermediate as well as complex cross-talks between different binding partners, implying a highly multifaceted conformational energy landscape of ßarr1 that can be intricately modulated during signaling.

Tailored Synthesis of Two Metal Borates KSrM3B2O9 (M = Al and Ga) Exhibiting Wide Ultraviolet Transparency.

Borate materials continue to command considerable attention due to their remarkable capacity for applications in deep ultraviolet (UV) wavelengths. Herein, two new metal borates KSrM3B2O9 (M = Al and Ga) were extracted via the application of flux techniques. These two crystals adopt a centrosymmetric space group P21/c (no. 14), showcasing a layered structural configuration composed of isolated [BO3] plane triangles and [AlO4]/[GaO4] tetrahedra. Thermal analysis revealed that KSrM3B2O9 (M = Al and Ga) exhibits an incongruent nature and possesses good thermal stability up to 1083 and 983 °C, respectively. Notably, these compounds display a short UV-transmission cutoff edge, approximately around 194 and 200 nm, accompanied by band gaps of 5.47 and 4.83 eV, respectively. Furthermore, KSrM3B2O9 (M = Al and Ga) demonstrates a moderate optical birefringence of 0.026 and 0.025, respectively. Additionally, first-principles calculations were employed to shed light on the intricate interplay between the structure and properties of these compounds.

Rational Design of a Niobium Tellurite Crystal Nb2Te3O11 Exhibiting Good Overall Infrared NLO Performance by Structural Genetic Engineering.

Nonlinear optical (NLO) materials have aroused increasing interest owing to their promising applications in optoelectronic technologies. Herein, we present the synthesis of an acentric niobium tellurite crystal, Nb2Te3O11, extracted via a spontaneous crystallization approach. It adopts a unique three-dimensional (3D) structure constructed by the distorted [TeO3], [TeO4], and [NbO6] fundamental building units. The title compound undergoes incongruent melting at approximately 807 °C. Optical characterizations demonstrate that Nb2Te3O11 possesses an extended transparency window beyond 5 µm, along with a large band gap value of 3.1 eV. Moreover, the as-synthesized Nb2Te3O11 displays an appreciable second-harmonic generation (SHG) response of 2 × KDP and a notable birefringence of 0.11 under 1064 nm for achieving phase-matching. In addition, theoretical calculation investigations suggest that the intriguing optical properties are ascribed to the cooperative effect of three types of NLO-active motifs: [TeO3] pyramids, [TeO4] seesaws, and [NbO6] octahedra. These attributes provide new functional insights into Nb2Te3O11 and enrich the family of NLO crystals in the mid-infrared region.

An NIR Fluorescence Turn-on and MRl Bimodal Probe for Concurrent Real-time in vivo Sensing and Labeling of ß-Galactosidase.

To realize sensing and labeling biomarkers is quite challenging in terms of designing multimodal imaging probes. In this study, we developed a novel ß-galactosidase (ß-gal) activated bimodal imaging probe that combines near-infrared (NIR) fluorescence and magnetic resonance imaging (MRI) to enable real-time visualization of activity in living organisms. Upon ß-gal activation, Gal-Cy-Gd-1 exhibits a remarkable 42-fold increase in NIR fluorescence intensity at 717 nm, allowing covalent labeling of adjacent target enzymes or proteins and avoiding molecular escape to promote probe accumulation at the tumor site. This fluorescence reaction enhances the longitudinal relaxivity by approximately 1.9 times, facilitating high-resolution MRI. The unique features of Gal-Cy-Gd-1 enable real-time and precise visualization of ß-gal activity in live tumor cells and mice. The probe's utilization aids in identifying in situ ovarian tumors, offering valuable assistance in the precise removal of tumor tissue during surgical procedures in mice. The fusion of NIR fluorescence and MRI activation through self-immobilizing target enzymes or proteins provides a robust approach for visualizing ß-gal activity. Moreover, this approach sets the groundwork for developing other activatable bimodal probes, allowing real-time in vivo imaging of enzyme activity and localization.

Electrocatalytic Urea Synthesis with 63.5 % Faradaic Efficiency and 100 % N-Selectivity via One-step C-N coupling.

Electrocatalytic urea synthesis via coupling N2 and CO2 provides an effective route to mitigate energy crisis and close carbon footprint. However, the difficulty on breaking N≡N is the main reason that caused low efficiencies for both electrocatalytic NH3 and urea synthesis, which is the bottleneck restricting their industrial applications. Herein, a new mechanism to overcome the inert of the nitrogen molecule was proposed by elongating N≡N instead of breaking N≡N to realize one-step C-N coupling in the process for urea production. We constructed a Zn-Mn diatomic catalyst with axial chloride coordination, Zn-Mn sites display high tolerance to CO poisoning and the Faradaic efficiency can even be increased to 63.5 %, which is the highest value that has ever been reported. More importantly, negligible N≡N bond breakage effectively avoids the generation of ammonia as intermediates, therefore, the N-selectivity in the co-electrocatalytic system reaches100 % for urea synthesis. The previous cognition that electrocatalysts for urea synthesis must possess ammonia synthesis activity has been broken. Isotope-labelled measurements and Operando synchrotron-radiation Fourier transform infrared spectroscopy validate that activation of N-N triple bond and nitrogen fixation activity arise from the one-step C-N coupling process of CO species with adsorbed N2 molecules.

Structural basis for high-affinity recognition of aflatoxin B1 by a DNA aptamer.

The 26-mer DNA aptamer (AF26) that specifically binds aflatoxin B1 (AFB1) with nM-level high affinity is rare among hundreds of aptamers for small molecules. Despite its predicted stem-loop structure, the molecular basis of its high-affinity recognition of AFB1 remains unknown. Here, we present the first high-resolution nuclear magnetic resonance structure of AFB1-AF26 aptamer complex in solution. AFB1 binds to the 16-residue loop region of the aptamer, inducing it to fold into a compact structure through the assembly of two bulges and one hairpin structure. AFB1 is tightly enclosed within a cavity formed by the bulges and hairpin, held in a place between the G·C base pair, G·G·C triple and multiple T bases, mainly through strong π-π stacking, hydrophobic and donor atom-π interactions, respectively. We further revealed the mechanism of the aptamer in recognizing AFB1 and its analogue AFG1 with only one-atom difference and introduced a single base mutation at the binding site of the aptamer to increase the discrimination between AFB1 and AFG1 based on the structural insights. This research provides an important structural basis for understanding high-affinity recognition of the aptamer, and for further aptamer engineering, modification and applications.