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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. [ABSTRACT FROM AUTHOR]

β-Arrestins (βarrs) 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 βarr activation, emerging evidence highlights the contribution from membrane lipids. However, detailed molecular mechanisms of βarr activation by different binding partners remain elusive. In this work, we present a comprehensive study of the structural changes in critical regions of βarr1 during activation using 19F NMR spectroscopy. We show that phosphopeptides derived from different classes of GPCRs display different βarr1 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 βarr1 that can be intricately modulated during signaling. The molecular basis for the functional versatility of β-arrestins in the G-protein coupled receptor signaling pathway is not well understood. Here, the authors use19F NMR spectroscopy to show that different binding partners activate β-arrestin-1 through distinct mechanisms. [ABSTRACT FROM AUTHOR]

In‐cell NMR spectroscopy is an effective tool for observing proteins at atomic resolution in their native cellular environment. However, its utility is limited by its low sensitivity and the extensive line broadening caused by nonspecific interactions in the cells, which is even more pronounced in human cells due to the difficulty of overexpressing or delivering high concentrations of isotopically labeled proteins. Here, we present a high‐sensitivity tag (wPSP‐6F) containing two trifluoromethyl groups that can efficiently label globular proteins with molecular weights in the 6–40 kDa range under mild conditions. This tag allowed us to detect globular proteins in human cells at concentrations as low as 1.0 μM, which would not have been achievable with 15N or 3‐fluorotyrosine labeling. Moreover, we detected conformational changes and interactions of proteins in the cellular environment. The new sensitive 19F NMR tag may significantly expand the scope of protein NMR in human cells. [ABSTRACT FROM AUTHOR]

α‐Amylase, an essential biomarker in pancreas related diseases and perform a major role in carbohydrates metabolism. Hence, monitoring the dynamic changes of α‐amylase is crucial for better clinical diagnosis of diseases. However, the existing methods are suffered from low sensitivity, time consumption and indirect assay with aid of tool enzyme or inhibitor of competitive substrates, the rapid and non‐destructive sensing of α‐amylase in biological samples was highly desired. In this work, a very simple tetraphenylethylene motif and γ‐cyclodextrin based supramolecular fluometric sensing system was firstly established. This system has no emission signal in aqueous media for the freely rotation of phenyl rings in the cavity of γ‐cyclodextrin, but the AIE residues can be released in to water after the α‐amylase hydrolysing γ‐cyclodextrin, then turn on the fluorescence. In this system, the detection limit is calculated to be 0.007 U mL−1 in MES buffer with a linear range of 0–0.35 U mL−1, having excellent selectivity to α‐amylase compared to other proteins. At last, our probe can be applied to the quantitative analysis of α‐amylase in human serum, showing potential in point of care testing. How to be a good guest: A supramolecular probe based on the tetraphenylethylene motif and γ‐cyclodextrin host–guest inclusion for α‐amylase recognition was designed and synthesized. The specific cleavage of the 1,4‐glycosidic bonds in cyclodextrin by α‐amylase results in the release of the guest molecule TPESO3Na, a relatively amphiphilic moiety that self‐assembles into a highly fluorescent micelle, thus giving rise to "turn‐on" fluorescent signals. [ABSTRACT FROM AUTHOR]

A tetraaryl‐1,4‐dihydropyrrolo‐[3,2‐b]pyrroles (TAPP) moiety with the combination of two pyrrole rings and four phenyl moieties demonstrated strong electron‐donating ability and nonplanar configuration simultaneously. Once incorporated into the organic dyes as a novel electron donor, it was beneficial for the enhancement of light‐harvesting ability and suppression of electron recombination in the photovoltaic and photocatalysis systems. With the linkage of tunable conjugated bridges and electron acceptor, the corresponding organic dyes exhibited improved photovoltaic performance in dye‐sensitized solar cells and facilitated photocatalytic hydrogen generation with a highest turnover number (TON) of 4337. Through the detailed investigation of relationship between molecular structures and photovoltaic/photocatalysis property, the connection and difference in molecular design for these two systems are well explained, with the aim to promote the application of dye‐sensitized technology in various fields. TAPP dance: With the incorporation of tetraaryl‐1,4‐dihydropyrrolo‐[3,2‐b]pyrroles (TAPP) derivatives into organic dyes as novel electron donors, the optimized photovoltaic and photocatalysis performance is achieved by the efficient suppression of electron recombination in different conditions. [ABSTRACT FROM AUTHOR]

In vitro, 19 F 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 19 F 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., (© 2024 Wiley‐VCH GmbH.)

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., (© 2024 Wiley-VCH GmbH.)

In-cell NMR spectroscopy is an effective tool for observing proteins at atomic resolution in their native cellular environment. However, its utility is limited by its low sensitivity and the extensive line broadening caused by nonspecific interactions in the cells, which is even more pronounced in human cells due to the difficulty of overexpressing or delivering high concentrations of isotopically labeled proteins. Here, we present a high-sensitivity tag (wPSP-6F) containing two trifluoromethyl groups that can efficiently label globular proteins with molecular weights in the 6-40 kDa range under mild conditions. This tag allowed us to detect globular proteins in human cells at concentrations as low as 1.0 μM, which would not have been achievable with 15 N or 3-fluorotyrosine labeling. Moreover, we detected conformational changes and interactions of proteins in the cellular environment. The new sensitive 19 F NMR tag may significantly expand the scope of protein NMR in human cells., (© 2023 Wiley-VCH GmbH.)

Thiols play critical roles in regulating biological functions and have wide applications in pharmaceutical and biomedical industries. However, we still lack a general approach for the simultaneous detection of various thiols, especially in complex systems. Herein, we establish a 19 F NMR platform where thiols are selectively fused into a novelly designed fluorinated receptor that has two sets of environmentally different 19 F atoms with fast kinetics ( k 2 = 0.73 mM -1 min -1 ), allowing us to generate unique two-dimensional codes for about 20 thiols. We demonstrate the feasibility of the approach by reliably quantifying thiol drug content in tablets, discriminating thiols in living cells, and for the first time monitoring the thiol related metabolism pathway at the atomic level. Moreover, the method can be easily extended to detect the activity of thiol related enzymes such as γ-glutamyl transpeptidase. We envision that the versatile platform will be a useful tool for detecting thiols and elucidating thiol-related processes in complex systems., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

α-Amylase, an essential biomarker in pancreas related diseases and perform a major role in carbohydrates metabolism. Hence, monitoring the dynamic changes of α-amylase is crucial for better clinical diagnosis of diseases. However, the existing methods are suffered from low sensitivity, time consumption and indirect assay with aid of tool enzyme or inhibitor of competitive substrates, the rapid and non-destructive sensing of α-amylase in biological samples was highly desired. In this work, a very simple tetraphenylethylene motif and γ-cyclodextrin based supramolecular fluometric sensing system was firstly established. This system has no emission signal in aqueous media for the freely rotation of phenyl rings in the cavity of γ-cyclodextrin, but the AIE residues can be released in to water after the α-amylase hydrolysing γ-cyclodextrin, then turn on the fluorescence. In this system, the detection limit is calculated to be 0.007 U mL -1 in MES buffer with a linear range of 0-0.35 U mL -1 , having excellent selectivity to α-amylase compared to other proteins. At last, our probe can be applied to the quantitative analysis of α-amylase in human serum, showing potential in point of care testing., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A novel ladderlike fused-ring donor, dithienocyclopentacarbazole (DTCC) derivative, is used to design and synthesize three novel donor-acceptor-π-acceptor-type organic dyes (C1-C3) via facile direct arylation reactions, in which the DTCC derivative substituted by four p -octyloxyphenyl groups is served as the electron donor and the carboxylic acid group is used as the electron acceptor or anchoring group. To fine-tune the optical, electrochemical, and photovoltaic properties of the three dyes, various auxiliary acceptors, including benzo[2,1,3]thiadiazole (BT), 5,6-difluorobenzo[2,1,3]thiadiazole (DFBT), and pyridal[2,1,3]thiadiazole (PT), are incorporated into the dye backbones. The results indicate that all of the three dyes exhibit strong light-capturing ability in the visible region and obtain relatively high molar extinction coefficients (>31 000 M -1 cm -1 ) due to their strong charge transfer (CT) from donor to acceptor. Moreover, theoretical model calculations demonstrate fully separated highest occupied molecular orbital and lowest unoccupied molecular orbital energy levels for the three dyes, which is helpful for efficient charge separation and electron injection. Using the three dyes as sensitizers, conventional dye-sensitized solar cells (DSSCs) based on liquid iodide/triiodide redox electrolytes are fabricated. Our results indicate that the BT-containing dye C1 affords the highest power conversion efficiency of up to 6.75%, much higher than that of the DFBT-containing dye C2 (5.40%) and the PT-containing dye C3 (1.85%). To our knowledge, this is the first example reported in the literature where the DTCC unit has been used to develop novel organic dyes for DSSC applications. Our work unambiguously demonstrates that the ladderlike DTCC derivatives are the superb electron-donating blocks for the development of high-performance organic dyes., Competing Interests: The authors declare no competing financial interest.