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Microbial tryptophan (Trp) metabolites acting as aryl hydrocarbon receptor (AhR) ligands are shown to effectively improve metabolic diseases via regulating microbial community. However, the underlying mechanisms by which Trp metabolites ameliorate bone loss via gut‐bone crosstalk are largely unknown. In this study, supplementation with Trp metabolites, indole acetic acid (IAA), and indole‐3‐propionic acid (IPA), markedly ameliorate bone loss by repairing intestinal barrier integrity in ovariectomy (OVX)‐induced postmenopausal osteoporosis mice in an AhR‐dependent manner. Mechanistically, intestinal AhR activation by Trp metabolites, especially IAA, effectively repairs intestinal barrier function by stimulating Wnt/β‐catenin signaling pathway. Consequently, enhanced M2 macrophage by supplementation with IAA and IPA secrete large amount of IL‐10 that expands from intestinal lamina propria to bone marrow, thereby simultaneously promoting osteoblastogenesis and inhibiting osteoclastogenesis in vivo and in vitro. Interestingly, supplementation with Trp metabolites exhibit negligible ameliorative effects on both gut homeostasis and bone loss of OVX mice with intestinal AhR knockout (VillinCreAhrfl/fl). These findings suggest that microbial Trp metabolites may be potential therapeutic candidates against osteoporosis via regulating AhR‐mediated gut‐bone axis. [ABSTRACT FROM AUTHOR]

Interactions between proteins and osmolytes are ubiquitous within cells, assisting in response to environmental stresses. However, our understanding of protein–osmolyte interactions underlying desiccation tolerance is limited. Here, we employ solid‐state NMR (ssNMR) to derive information about protein conformation and site‐specific interactions between the model protein, SH3, and the osmolyte trimethylamine N‐oxide (TMAO). The data show that SH3–TMAO interactions maintain key structured regions during desiccation and facilitate reversion to the protein's native state once desiccation stress is even slightly relieved. We identify 10 types of residues at 28 sites involved in the SH3–TMAO interactions. These sites comprise hydrophobic, positively charged, and aromatic amino acids located in SH3's hydrophobic core and surface clusters. TMAO locks both the hydrophobic core and surface clusters through its zwitterionic and trimethyl ends. This double locking is responsible for desiccation tolerance and differs from ideas based on exclusion, vitrification, and water replacement. ssNMR is a powerful tool for deepening our understanding of extremely weak protein−osmolyte interactions and providing insight into the evolutionary mechanism of environmental tolerance. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

To realize sensing and labeling biomarkers is quite challenging in terms of designing multimodal imaging probes. In this study, we developed a novel β‐galactosidase (β‐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 β‐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 β‐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 β‐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. [ABSTRACT FROM AUTHOR]

Protein dynamics play a crucial role in their diverse functions. The intracellular environment significantly influences protein dynamics, particularly for intrinsically disordered proteins (IDPs). To comprehensively capture structural information from various proteins within cells and characterize protein dynamics, chemical cross‐linking mass spectrometry was employed. In this study, we introduce a hierarchical decoding strategy that enables the investigation of protein dynamics in vivo. Computational analysis based on distance restraints derived from cross‐links is used to infer protein dynamics in cells. To facilitate this analysis, we leverage the prior structure obtained from AlphaFold2. By employing this strategy, we can characterize the full‐length structure of multi‐domain proteins taking into account their distinct dynamic features. Furthermore, by combining restraint sampling with an unbiased sampling and evaluation approach, we can provide a comprehensive description of the intrinsic motion of IDPs. Consequently, the hierarchical strategy we propose holds significant potential in advancing our understanding of the molecular mechanisms that undelie protein functions in cells. [ABSTRACT FROM AUTHOR]

The aggregation of amyloidogenic proteins is often related to the occurrence of neurodegenerative diseases, including fused in sarcoma protein (FUS) in frontotemporal lobar degeneration and amyotrophic lateral sclerosis diseases. Recently, the SERF protein family has been reported to have a significant regulatory effect on amyloid formation, but it is still unclear about the detailed mechanisms of SERF acting on different amyloidogenic proteins. Herein, nuclear magnetic resonance (NMR) spectroscopy and fluorescence spectroscopy were used to explore interactions of ScSERF with three amyloidogenic proteins FUS‐LC, FUS‐Core, and α‐Synuclein. NMR chemical shift perturbations reveal them sharing similar interaction sites on the N‐terminal region of ScSERF. However, the amyloid formation of α‐Synuclein protein is accelerated by ScSERF, while ScSERF inhibits fibrosis of FUS‐Core and FUS‐LC proteins. Both the primary nucleation and the total amount of fibrils produced are detained. Our results suggest a diverse role of ScSERF in regulating the fibril growth of amyloidogenic proteins. [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]

Single cell analysis has drawn increasing interest from the research community due to its capability to interrogate cellular heterogeneity, allowing refined tissue classification and facilitating novel biomarker discovery. With the advancement of relevant instruments and techniques, it is now possible to perform multiple omics including genomics, transcriptomics, metabolomics or even proteomics at single cell level. In comparison with other omics studies, single‐cell metabolomics (SCM) represents a significant challenge since it involves many types of dynamically changing compounds with a wide range of concentrations. In addition, metabolites cannot be amplified. Although difficult, considerable progress has been made over the past decade in mass spectrometry (MS)‐based SCM in terms of processing technologies and biochemical applications. In this review, we will summarize recent progress in the development of promising MS platforms, sample preparation methods and SCM analysis of various cell types (including plant cell, cancer cell, neuron, embryo cell, and yeast cell). Current limitations and future research directions in the field of SCM will also be discussed. [ABSTRACT FROM AUTHOR]

Nitroreductase (NTR) is an important biomarker widely used to evaluate the degree of tumor hypoxia. Although a few optical methods have been reported for detecting nitroreductase at low concentration ranges, an effective strategy for nitroreductase monitoring in vivo without limits to the imaging depth is still lacking. Herein, a novel dual‐mode NIR fluorescence and 19F MRI agent, FCy7‐NO2, is proposed for imaging tumor hypoxia. We show that FCy7‐NO2 serves as not only a rapid NIR fluorescence enhanced probe for monitoring and bioimaging of nitroreductase in tumors, but also a novel 19F MR chemical shift‐sensitive contrast agent for selectively detecting nitroreductase catalyzed reduction. Notably, integrating two complementary imaging technologies into FCy7‐NO2 enables sensitive detection of nitroreductase in a broad concentration range without tissue‐depth limit. In general, this agent has a remarkable response to nitroreductase, which provides a promising method for understanding tumor evolution and its physiological role in the hypoxic microenvironment. [ABSTRACT FROM AUTHOR]

Molecular chaperones are diverse families of proteins that play key roles in protein homeostasis. They assist the folding of client proteins or prevent them from irreversible aggregation under stress conditions. Diverse chaperone families contribute to different aspects of protein homeostasis by interacting with a wide range of client proteins. Despite the vital roles of chaperones in cell survival, the molecular mechanisms underlying chaperone functions remain elusive, due to the non‐specificity of chaperone‐client interactions and the intrinsic flexibility of the clients. Our understanding of the chaperone functional mechanisms, especially regarding chaperone‐client interactions, has greatly expanded in recent years, thanks to the significant contribution from various NMR studies. Solution NMR methods have unique advantages in characterizing disordered protein structures, detecting weak and non‐specific interactions, and probing conformational dynamics of proteins and protein complexes, etc., and therefore are especially powerful in the studies of chaperone structure‐function relationships. In this review, we summarize some of the current knowledge of molecular chaperones, with emphasis on common features of chaperone‐client interactions and examples on a number of specific systems in which solution NMR methods were used to provide essential insights into their functional mechanisms. [ABSTRACT FROM AUTHOR]

Protein CGL2373 from Corynebacterium glutamicum was previously proposed to be a member of the polyketide_cyc2 family, based on amino‐acid sequence and secondary structure features derived from NMR chemical shift assignments. We report here the solution NMR structure of CGL2373, which contains three α‐helices and one antiparallel β‐sheet and adopts a helix‐grip fold. This structure shows moderate similarities to the representative polyketide cyclases, TcmN, WhiE, and ZhuI. Nevertheless, unlike the structures of these homologs, CGL2373 structure looks like a half‐open shell with a much larger pocket, and key residues in the representative polyketide cyclases for binding substrate and catalyzing aromatic ring formation are replaced with different residues in CGL2373. Also, the gene cluster where the CGL2373‐encoding gene is located in C. glutamicum contains additional genes encoding nucleoside diphosphate kinase, folylpolyglutamate synthase, and valine‐tRNA ligase, different from the typical gene cluster encoding polyketide cyclase in Streptomyces. Thus, although CGL2373 is structurally a polyketide cyclase‐like protein, the function of CGL2373 may differ from the known polyketide cyclases and needs to be further investigated. The solution structure of CGL2373 lays a foundation for in silico ligand screening and binding site identifying in future functional study. [ABSTRACT FROM AUTHOR]

Purpose: CEST has become a preeminent technology for the rapid detection and grading of tumors, securing its widespread use in both laboratory and clinical research. However, many existing CEST MRI agents exhibit a sensitivity limitation due to small chemical shifts between their exchangeable protons and water. We propose a new group of CEST MRI agents, free‐base porphyrins and chlorin, with large exchangeable proton chemical shifts from water for enhanced detection. Methods: To test these newly identified CEST agents, we acquired a series of Z‐spectra at multiple pH values and saturation field strengths to determine their CEST properties. The data were analyzed using the quantifying exchange using saturation power method to quantify exchange rates. After identifying several promising candidates, a porphyrin solution was injected into tumor‐bearing mice, and MR images were acquired to assess detection feasibility in vivo. Results: Based on the Z‐spectra, the inner nitrogen protons in free‐base porphyrins and chlorin resonate from −8 to −13.5 ppm from water, far shifted from the majority of endogenous metabolites (0‐4 ppm) and Nuclear Overhauser enhancements (−1 to −3.5 ppm) and far removed from the salicylates, imidazoles, and anthranillates (5‐12 ppm). The exchange rates are sufficiently slow to intermediate (500‐9000 s−1) to allow robust detection and were sensitive to substituents on the porphyrin ring. Conclusion: These results highlight the capabilities of free‐base porphyrins and chlorin as highly upfield CEST MRI agents and provide a new scaffold that can be integrated into a variety of diagnostic or theranostic agents for biomedical applications. [ABSTRACT FROM AUTHOR]

We report the solution nuclear magnetic resonance (NMR) structure of CHU_1110 from Cytophaga hutchinsonii. CHU_1110 contains three α‐helices and one antiparallel β‐sheet, forming a large cavity in the center of the protein, which are consistent with the structural characteristics of AHSA1 protein family. This protein shows high structural similarities to the prokaryotic proteins RHE_CH02687 from Rhizobium etli and YndB from Bacillussubtilis, which can bind with flavinoids. Unlike these two homologs, CHU_1110 shows no obvious interaction with flavonoids in NMR titration experiments. In addition, no direct interaction has been observed between CHU_1110 and ATP, although many homologous sequences of CHU_1110 have been annotated as ATPase. Combining the analysis of structural similarity of CHU_1110 and genomic context of its encoding gene, we speculate that CHU_1110 may be involved in the stress response of bacteria to heavy metal ions, even though its specific biological functions that need to be further investigated. [ABSTRACT FROM AUTHOR]

A new magnetic resonance imaging (MRI) molecular sensor for hydrogen sulfide detection and imaging using the nuclear spin resonance of hyperpolarized 129Xe is developed. The designed MRI sensor employs cryptophane for NMR sensing, together with an azide group serving as a reaction site. It demonstrates a 'proof-of-concept' that a fluorescent H2S probe can be linked to a xenon-binding cryptophane and thereby converted into an MRI probe, which could provide a very generalizable template. [ABSTRACT FROM AUTHOR]

Macromolecular crowding and confinement are two factors that potentially affect protein structure and function in a complex cellular environment. The confinement effect on the structure and function of holoCaM [Ca2+-loaded calmodulin (CaM)], a two-domain protein involved in many calcium-mediated signaling pathways, has been investigated previously. However, little is known about how macromolecular crowding affects holoCaM structure and function. Here, the structure-function correlations of holoCaM are investigated in macromolecular crowded environments. It was found that macromolecular crowding impacts its structure and function mildly. The major conformational states are still extended conformation with inter-domain separation in crowded environment as well as those in dilute solution, but the population of transiently compact conformation increases compared to dilute solution. Furthermore, macromolecular crowding facilitates the binding of CaM with AcN19 peptide (CaM-bind domain of α- syn). This study provides a direct comparison for macromolecular crowding and confinement effects on protein structure and function, which helps to understand chemistry regulation in living cells. [ABSTRACT FROM AUTHOR]

ABSTRACT We report the solution NMR structure of RHE_CH02687 from Rhizobium etli. Its structure consists of two β-sheets that together with two short and one long α-helix form a hydrophobic cavity. This protein shows a high structural similarity to the prokaryotic protein YndB from Bacillus subtilis, and the eukaryotic protein Aha1. NMR titration experiments confirmed that RHE_CH02687, like its homolog YndB, interacted with flavonoids, giving support for a biological function as a flavonoid sensor in the symbiotic interaction between R. etli and plants. In addition, our study showed no evidence for a direct interaction between RHE_CH02687 and HtpG, the R. etli homolog of Hsp90. Proteins 2017; 85:951-956. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

ABSTRACT Human INSM1 containing five C-terminal C2H2-type zinc fingers (ZFs), is a key regulator of neuroendocrine development. Previous research reported that full-length INSM1 containing all five ZFs recognized a consensus DNA sequence. Structure elucidation of human INSM1 ZFs is currently insufficient to understand the DNA binding mechanism. Herein, we present the solution NMR structure of ZF4-5, in which the two ZFs adopt a head-to-tail arrangement and each ZF features a canonical ββα fold. NMR titrations and isothermal titration calorimetry experiments showed that ZF4-5 binds weakly to the consensus DNA sequence. Proteins 2017; 85:957-962. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Many cellular reactions involving proteins, including their biosynthesis, misfolding, and transport, occur in confined compartments. Despite its importance, a structural basis of understanding of how confined environments alter protein function is still lacking. Herein, we explore structure-function correlations of calmodulin (CaM), a multidomain protein involved in many calcium-mediated signaling pathways, in reverse micelles. Confinement dramatically alters CaM structure and function. The protein forms an extended structure in bulk water, but becomes compacted in reverse micelles. In addition, confinement changes the function of CaM. Specifically, the protein binds the MLCK, AcN19, and somatostatin peptides in dilute buffer, but binds only the MLCK and AcN19 peptides in reverse micelles. In summary, we determined a new CaM structure in reverse micelles and demonstrate that confinement can modulate both protein structure and function. [ABSTRACT FROM AUTHOR]

The glutamine binding protein (GlnBP) binds l-glutamine and cooperates with its cognate transporters during glutamine uptake. Crystal structure analysis has revealed an open and a closed conformation for apo- and holo-GlnBP, respectively. However, the detailed conformational dynamics have remained unclear. Herein, we combined NMR spectroscopy, MD simulations, and single-molecule FRET techniques to decipher the conformational dynamics of apo-GlnBP. The NMR residual dipolar couplings of apo-GlnBP were in good agreement with a MD-derived structure ensemble consisting of four metastable states. The open and closed conformations are the two major states. This four-state model was further validated by smFRET experiments and suggests the conformational selection mechanism in ligand recognition of GlnBP. [ABSTRACT FROM AUTHOR]

A holistic and comparative quality assessment of vinegars from different countries is needed with international trade of vinegar become frequent. In this study, compounds characterization and comparison of commercial-grade Chinese cereal and European grape vinegars were performed using 1H NMR spectroscopy coupled with principal component analysis (PCA) and orthogonal projection to latent structures discriminate analysis (OPLS-DA). The results showed that Balsamic vinegars of Modena were clearly discriminated by higher amount of fructose and glucose, while Chinese aromatic vinegar and aged vinegars were characterized by higher amount of amino acids, volatile compounds, succinate and betaine. On the other hand, flavoring compounds in Chinese rice vinegar and European wine vinegars are less than the others. These characteristic components are associated with the special raw materials and producing process of each types of vinegar and endow them special flavor. The results obtained in this study provide a global insight into vinegar through a 1H NMR based compounds analysis that allows a holistic quality assessment and comparison of vinegars from different manufacture origins. [ABSTRACT FROM AUTHOR]

A bimodal imaging probe FCy7‐NO2 was applied to image nitroreductase overexpressed by lung cancer in vivo, as reported by Xin Zhou and co‐workers in their Research Article (e202213495). Integrating two complementary imaging technologies into FCy7‐NO2 enables sensitive detection of nitroreductase over a broad concentration range without tissue‐depth limit. [ABSTRACT FROM AUTHOR]

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Enhancement of images with weak edges faces great challenges in imaging applications. In this study, the authors propose a novel image enhancement approach based on intuitionistic fuzzy sets. The proposed method first divides an image into sub‐object and sub‐background areas, and then successively implements new fuzzification, hyperbolisation, and defuzzification operations on each area. In this way, an enhanced image is obtained, where the visual quality of region of interest (ROI) is significantly improved. Several types of images are utilised to validate the proposed method with respect to the enhancement performance. Experimental results demonstrate that the proposed algorithm not only works more stably for different types of images, but also has better enhancement performance, in comparison to conventional methods. This is a great merit of such design for discerning specific ROIs. [ABSTRACT FROM AUTHOR]

Mercury pollution, in the form of mercury ions (Hg2+), is a major health and environmental hazard. Commonly used sensors are invasive and limited to point measurements. Fluorescence-based sensors do not provide depth resolution needed to image spatial distributions. Herein we report a novel sensor capable of yielding spatial distributions by MRI using hyperpolarized 129Xe. A molecular clamp probe was developed consisting of dipyrrolylquinoxaline (DPQ) derivatives and twocryptophane-A cages. The DPQ derivatives act as cation receptors whereas cryptophane-A acts as a suitable host molecule for xenon. When the DPQ moiety interacts with mercury ions, the molecular clamp closes on the ion. Due to overlap of the electron clouds of the two cryptophane-A cages, the shielding effect on the encapsulated Xe becomes important. This leads to an upfield change of the chemical shift of the encapsulated Xe. This sensor exhibits good selectivity and sensitivity toward the mercury ion. This mercury-activated hyperpolarized 129Xe-based chemosensor is a new concept method for monitoring Hg2+ ion distributions by MRI. [ABSTRACT FROM AUTHOR]

We used Xenopus laevis oocytes, a paradigm for a variety of biological studies, as a eukaryotic model system for in-cell protein NMR spectroscopy. The small globular protein GB1 was one of the first studied in Xenopus oocytes, but there have been few reports since then of high-resolution spectra in oocytes. The scarcity of data is at least partly due to the lack of good labeling strategies and the paucity of information on resonance broadening mechanisms. Here, we systematically evaluate isotope enrichment and labeling methods in oocytes injected with five different proteins with molecular masses of 6 to 54 kDa. 19F labeling is more promising than 15N, 13C, and 2H enrichment. We also used 19F NMR spectroscopy to quantify the contribution of viscosity, weak interactions, and sample inhomogeneity to resonance broadening in cells. We found that the viscosity in oocytes is only about 1.2 times that of water, and that inhomogeneous broadening is a major factor in determining line width in these cells. [ABSTRACT FROM AUTHOR]

The influenza fusion peptide located at the N-terminus of the hemagglutinin HA2 subunit initiates the fusing process of the viral membrane with the host cell endosomal membrane. It had been reported that the structure of a 20-residue H3 subtype fusion peptide (H3-HAfp20) was significantly different with that of a H1 subtype 23-residue one (H1-HAfp23). The sequential difference between the 12th and 15th residues of H1 and H3 subtypes could not fully explain the conformational variation. The first and last three amino acids of H3-HAfp23 involved in formation of hydrogen bonds may play an important role in fusion process. To confirm this hypothesis, we investigate the structures of H3-HAfp23 peptide and its mutants, G1S and G1V, in dodecylphosphatidyl choline micelles by using heteronuclear NMR technology. The results demonstrate that, similar to H1-HAfp23 but significantly different with H3-HAfp20, H3-HAfp23 also has tight helical hairpin structure with the N- and C-terminuses linked together because of the hydrogen bonds between Gly1 and the last three amino acids, Trp21-Tyr22-Gly23. Although the 'hemifusion' G1S and lethal G1V mutants have hairpin-like helical structures, the distances between the N- and C-terminuses are increased as shortage of the hydrogen bonds and the larger kink angle between the antiparallel helices. The paramagnetic ion titration experiments show that the terminuses are inserted into the dodecylphosphatidyl choline micelles used as solving media. These may imply that the tight helical hairpin structure, especially the closed conformation at terminus, plays an important role in fusion activity. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Protein mobility in living cells is vital for cell function. Both cytosolic viscosity and weak protein-protein interactions affect mobility, but examining viscosity and weak interaction effects is challenging. Herein, we demonstrate the use of 19F NMR spectroscopy to measure cytoplasmic viscosity and to characterize nonspecific protein-protein interactions in living Escherichia coli cells. The origins of resonance broadening in Escherichia coli cells were also investigated. We found that sample inhomogeneity has a negligible effect on resonance broadening, the cytoplasmic viscosity is only about 2-3 times that of water, and ubiquitous transient weak protein-protein interactions in the cytosol play a significant role in governing the detection of proteins by using in-cell NMR spectroscopy. [ABSTRACT FROM AUTHOR]

Abstract: Most proteins function in cells where protein concentrations can reach 400g/l. However, most quantitative studies of protein properties are performed in idealized, dilute conditions. Recently developed in-cell NMR techniques can provide protein structure and other biophysical properties inside living cells at atomic resolution. Here we review how protein dynamics, including global and internal motions have been characterized by in-cell NMR, and then discuss the remaining challenges and future directions. [Copyright &y& Elsevier]

The Ca2+-mediated conformational transition of the protein calmodulin (CaM) is essential to a variety of signal transduction pathways. Whether the transition in living cells is similar to that observed in buffer is not known. Here, we report the direct observation by 19F NMR spectroscopy of the transition of the Ca2+-free and -bound forms in Xenopus laevis oocytes at different Ca2+ levels. We find that the Ca2+-bound CaM population increased greatly upon binding the target protein myosin light-chain kinase (MLCK) at the same Ca2+ level. Paramagnetic NMR spectroscopy was also exploited for the first time to obtain long-range structural constraints in cells. Our study shows that 19F NMR spectroscopy can be used to obtain long-range structural constraints in living eukaryotic cells and paves the way for quantification of protein binding constants. [ABSTRACT FROM AUTHOR]

The Ca2+-mediated conformational transition of the protein calmodulin (CaM) is essential to a variety of signal transduction pathways. Whether the transition in living cells is similar to that observed in buffer is not known. Here, we report the direct observation by 19F NMR spectroscopy of the transition of the Ca2+-free and -bound forms in Xenopus laevis oocytes at different Ca2+ levels. We find that the Ca2+-bound CaM population increased greatly upon binding the target protein myosin light-chain kinase (MLCK) at the same Ca2+ level. Paramagnetic NMR spectroscopy was also exploited for the first time to obtain long-range structural constraints in cells. Our study shows that 19F NMR spectroscopy can be used to obtain long-range structural constraints in living eukaryotic cells and paves the way for quantification of protein binding constants. [ABSTRACT FROM AUTHOR]

Hyperpolarization is one of the methods for NMR signal enhancement. NMR sensitivity can be further enhanced by combining hyperpolarization with chemical exchange saturation transfer (CEST), which termed “Hyper‐CEST”. A H2S sensor is reported that contains a cryptophane group for NMR sensing and a naphthalimide group for fluorescence sensing, together with an azide group as the reaction site. Upon reacting with H2S, the fluorescence and 129Xe NMR signal changes, which may provide a universal template for converting a fluorescence sensor into an 129Xe Hyper‐CEST MRI sensor. More information can be found in the Communication by X. Zhou, M. Liu, et al. on page 7648. [ABSTRACT FROM AUTHOR]

NMR Spectroscopy In their Communication on page 8686 ff., C. Li et al., demonstrate that 19F labeling is a good first choice for studying globular and disordered proteins in Xenopus oocytes, especially compared with conventional 15N ‐ or 13C ‐ methyl enrichment. By using 19F labeling, they found that, unlike E. coli cells, the viscosity in oocytes is only about 1.2 times that of water and that inhomogeneous broadening contributes 60 – 70 % to the line width. The labeling strategies and resonance broadening mechanisms in Xenopus oocytes were explored with the goal of expanding the application of this cell type. [ABSTRACT FROM AUTHOR]