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.

Visualizing Proteins in Human Cells at Near-Physiological Concentrations with Sensitive 19 F NMR Chemical Tags.

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.

Membrane-mediated disorder-to-order transition of SNAP25 flexible linker facilitates its interaction with syntaxin-1 and SNARE-complex assembly.

The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex comprises synaptosome-associated protein of 25 kDa (SNAP25), syntaxin-1a (syx-1), and synaptobrevin 2, which is essential for many physiologic processes requiring membrane fusion. Several studies imply that the loop region of SNAP25 plays important roles in SNARE-complex assembly. However, why and how the flexible loop facilitates the complex assembly remains poorly understood because it is purposely deleted in almost all structural studies. By using NMR spectroscopy and circular dichroism spectropolarimetry, we characterized SNAP25 structure and interactions with other SNAREs in aqueous buffer and in the membrane. We found that the N-terminal of the SNAP25 loop region binds with membrane, and this interaction induced a disorder-to-order conformational change of the loop, resulting in enhanced interaction between the C-terminal of the SNAP25 loop and syx-1. We further proved that SNARE-complex assembly efficiency decreased when we disrupted the electrostatic interaction between C-terminal of the SNAP25 loop and syx-1, suggesting that the SNAP25 loop region facilitates SNARE-complex assembly through promoting prefusion SNARE binary complex formation. Our work elucidates the role of the flexible loop and the membrane environment in SNARE-complex assembly at the residue level, which helps to understand membrane fusion, a fundamental transport and communication process in cells.-Jiang, X., Zhang, Z., Cheng, K., Wu, Q., Jiang, L., Pielak, G. J., Liu, M., Li, C. Membrane-mediated disorder-to-order transition of SNAP25 flexible linker facilitates its interaction with syntaxin-1 and SNARE-complex assembly.

Crowding and Confinement Can Oppositely Affect Protein Stability.

Proteins encounter crowded and confined macromolecular milieus in living cells. Simple theory predicts that both environments entropically stabilize proteins if only hard-core repulsive interactions are considered. Recent studies show that chemical interactions between the surroundings and the test protein also play key roles such that the overall effect of crowding or confinement is a balance of hard-core repulsions and chemical interactions. There are, however, few quantitative studies. Here, we quantify the effects of crowding and confinement on the equilibrium unfolding thermodynamics of a model globular protein, KH1. The results do not agree with predictions from simple theory. KH1 is stabilized by synthetic-polymer crowding agents but destabilized by confinement in reverse micelles. KH1 is more entropically stabilized and enthalpically destabilized in concentrated solutions of the monomers than it is in solutions of the corresponding polymers. When KH1 is confined in reverse micelles, the temperature of maximum stability decreases, the melting temperature decreases, and the protein is entropically destabilized and enthalpically stabilized. Our results show the importance of chemical interactions to protein folding thermodynamics and imply that cells utilize chemical interactions to tune protein stability.

Ca(2+) modulating α-synuclein membrane transient interactions revealed by solution NMR spectroscopy.

α-Synuclein is involved in Parkinson's disease and its interaction with cell membrane is crucial to its pathological and physiological functions. Membrane properties, such as curvature and lipid composition, have been shown to affect the interactions by various techniques, but ion effects on α-synuclein membrane interactions remain elusive. Ca(2+) dynamic fluctuation in neurons plays important roles in the onset of Parkinson's disease and its influx is considered as one of the reasons to cause cell death. Using solution Nuclear Magnetic Resonance (NMR) spectroscopy, here we show that Ca(2+) can modulate α-synuclein membrane interactions through competitive binding to anionic lipids, resulting in dissociation of α-synuclein from membranes. These results suggest a negative modulatory effect of Ca(2+) on membrane mediated normal function of α-synuclein, which may provide a clue, to their dysfunction in neurodegenerative disease.

Cotton (Gossypium hirsutum) 14-3-3 proteins participate in regulation of fibre initiation and elongation by modulating brassinosteroid signalling.

Cotton (Gossypium hirsutum) fibre is an important natural raw material for textile industry in the world. Understanding the molecular mechanism of fibre development is important for the development of future cotton varieties with superior fibre quality. In this study, overexpression of Gh14-3-3L in cotton promoted fibre elongation, leading to an increase in mature fibre length. In contrast, suppression of expression of Gh14-3-3L, Gh14-3-3e and Gh14-3-3h in cotton slowed down fibre initiation and elongation. As a result, the mature fibres of the Gh14-3-3 RNAi transgenic plants were significantly shorter than those of wild type. This 'short fibre' phenotype of the 14-3-3 RNAi cotton could be partially rescued by application of 2,4-epibrassinolide (BL). Expression levels of the BR-related and fibre-related genes were altered in the Gh14-3-3 transgenic fibres. Furthermore, we identified Gh14-3-3 interacting proteins (including GhBZR1) in cotton. Site mutation assay revealed that Ser163 in GhBZR1 and Lys51/56/53 in Gh14-3-3L/e/h were required for Gh14-3-3-GhBZR1 interaction. Nuclear localization of GhBZR1 protein was induced by BR, and phosphorylation of GhBZR1 by GhBIN2 kinase was helpful for its binding to Gh14-3-3 proteins. Additionally, 14-3-3-regulated GhBZR1 protein may directly bind to GhXTH1 and GhEXP promoters to regulate gene expression for responding rapid fibre elongation. These results suggested that Gh14-3-3 proteins may be involved in regulating fibre initiation and elongation through their interacting with GhBZR1 to modulate BR signalling. Thus, our study provides the candidate intrinsic genes for improving fibre yield and quality by genetic manipulation.

Labeling strategy and signal broadening mechanism of Protein NMR spectroscopy in Xenopus laevis oocytes.

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. (19) F labeling is more promising than (15) N, (13) C, and (2) H enrichment. We also used (19) F 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.

Recent advances in protein NMR spectroscopy and their implications in protein therapeutics research.

Nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography are the two main methods for protein three-dimensional structure determination at atomic resolution. According to the protein structures deposited in the Protein Data Bank, X-ray crystallography has become the dominant method for structure determination, particularly for large proteins and complexes. However, with the developments of isotope labeling, increase of magnetic field strength, common use of a cryogenic probe, and ingenious pulse sequence design, the applications of NMR spectroscopy have expanded in biological research, especially in characterizing protein dynamics, sparsely populated transient structures, weak protein interactions, and proteins in living cells at atomic resolution, which is difficult if not impossible by other biophysical methods. Although great advances have been made in protein NMR spectroscopy, its applications in protein therapeutics, which represents the fastest growing segment of the pharmaceutical industry, are still limited. Here we review the recent advances in the use of NMR spectroscopy in studies of large proteins or complexes, posttranslation modifications, weak interactions, and aggregation, and in-cell NMR spectroscopy. The potential applications of NMR spectroscopy in protein therapeutic assays are discussed.

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.

(19) F NMR spectroscopy as a probe of cytoplasmic viscosity and weak protein interactions in living cells.

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 (19) F 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.

C-terminal truncation modulates α-Synuclein's cytotoxicity and aggregation by promoting the interactions with membrane and chaperone.

α-Synuclein (α-syn) is the main protein component of Lewy bodies, the major pathological hallmarks of Parkinson's disease (PD). C-terminally truncated α-syn is found in the brain of PD patients, reduces cell viability and tends to form fibrils. Nevertheless, little is known about the mechanisms underlying the role of C-terminal truncation on the cytotoxicity and aggregation of α-syn. Here, we use nuclear magnetic resonance spectroscopy to show that the truncation alters α-syn conformation, resulting in an attractive interaction of the N-terminus with membranes and molecular chaperone, protein disulfide isomerase (PDI). The truncated protein is more toxic to mitochondria than full-length protein and diminishes the effect of PDI on α-syn fibrillation. Our findings reveal a modulatory role for the C-terminus in the cytotoxicity and aggregation of α-syn by interfering with the N-terminus binding to membranes and chaperone, and provide a molecular basis for the pathological role of C-terminal truncation in PD pathogenesis.

A cotton gene encoding novel MADS-box protein is preferentially expressed in fibers and functions in cell elongation.

Cotton fibers, as natural fibers, are widely used in the textile industry in the world. In order to find genes involved in fiber development, a cDNA (designated as GhMADS11) encoding a novel MADS protein with 151 amino acid residues was isolated from cotton fiber cDNA library. The deduced protein shares high similarity with Arabidopsis AP1 and AGL8 in MADS domain. However, the GhMADS11 protein (being absent of the partial K-domain and normal C-terminus) is shorter than AP1 and AGL8 by the reason of gene frameshift mutation during evolution. The experimental results revealed that GhMADS11 was not a transcriptional activator, and it did not form homodimer. GhMADS11 transcripts were specifically accumulated in elongating fibers, but no or very low signals of its expression were detected in other tissues of cotton. Overexpression of GhMADS11 in fission yeast promotes atypical cell elongation by 1.4-2.0-fold. Furthermore, morphological analysis indicated that the transformed cells expressing GhMADS11m, a MIKC-type derivative of GhMADS11 by the site-directed mutation, displayed the same phenotype as that of the transformed cells with GhMADS11. The concurrence of these data sets suggested that GhMADS11 protein may function in fiber cell elongation, and its MADS domain and partial K-domain are sufficient for this function.

Two cotton Cys2/His2-type zinc-finger proteins, GhDi19-1 and GhDi19-2, are involved in plant response to salt/drought stress and abscisic acid signaling.

Cotton (Gossypium hirsutum) often encounters abiotic stress such as drought and high salinity during its development, and its productivity is significantly limited by those adverse factors. To investigate the molecular adaptation mechanisms of this plant species to abiotic stress, we identified two genes encoding Di19-like Cys2/His2 zinc-finger proteins in cotton. GFP fluorescence assay demonstrated that GhDi19-1 and GhDi19-2 are two nuclear-localized proteins. Quantitative RT-PCR and Northern blot analyses revealed that mRNA accumulation of both GhDi19-1 and GhDi19-2 was significantly promoted by salinity and drought. Expression of GUS gene driven by the GhDi19-1 and GhDi19-2 promoters, respectively, was intensively induced in cotyledons under NaCl and mannitol stresses. Overexpression of GhDi19-1 and GhDi19-2 in Arabidopsis resulted in the seedlings displaying hypersensitivity to high salinity and abscisic acid (ABA). Seed germination and seedling growth of the transgenic Arabidopsis were dramatically inhibited by salinity and ABA, compared with wild type. In addition, expression levels of the ABA-responsive genes ABF3, ABF4, ABI5 and KIN1 were also remarkably altered in the transgenic plants under ABA treatment. Collectively, our results suggested that both GhDi19-1 and GhDi19-2 may be involved in response to salt/drought stress and ABA signaling during early stages of plant development.

Interactome analysis of the six cotton 14-3-3s that are preferentially expressed in fibres and involved in cell elongation.

Proteins of the 14-3-3 family regulate a divergent set of signalling pathways in all eukaryotic organisms. In this study, several cDNAs encoding 14-3-3 proteins were isolated from a cotton fibre cDNA library. The Gh14-3-3 genes share high sequence homology at the nucleotide level in the coding region and at the amino acid level. Real-time quantitative RT-PCR analysis indicated that the expression of these Gh14-3-3 genes is developmentally regulated in fibres, and reached their peak at the stage of rapid cell elongation of fibre development. Furthermore, overexpression of Gh14-3-3a, Gh14-3-3e, and Gh14-3-3L in fission yeast promoted atypical longitudinal growth of the host cells. Yeast two-hybrid analysis revealed that the interaction between cotton 14-3-3 proteins is isoform selective. Through yeast two-hybrid screening, 38 novel interaction partners of the six 14-3-3 proteins (Gh14-3-3a, Gh14-3-3e, Gh14-3-3f, Gh14-3-3g, Gh14-3-3h, and Gh14-3-3L), which are involved in plant development, metabolism, signalling transduction, and other cellular processes, were identified in cotton fibres. Taking these data together, it is proposed that the Gh14-3-3 proteins may participate in regulation of fibre cell elongation. Thus, the results of this study provide novel insights into the 14-3-3 signalling related to fibre development of cotton.

Three cotton genes preferentially expressed in flower tissues encode actin-depolymerizing factors which are involved in F-actin dynamics in cells.

To investigate whether the high expression levels of actin-depolymerizing factor genes are related to pollen development, three GhADF genes (cDNAs) were isolated and characterized in cotton. Among them, GhADF6 and GhADF8 were preferentially expressed in petals, whereas GhADF7 displayed the highest level of expression in anthers, revealing its anther specificity. The GhADF7 transcripts in anthers reached its peak value at flowering, suggesting that its expression is developmentally-regulated in anthers. The GhADF7 gene including the promoter region was isolated from the cotton genome. To demonstrate the specificity of the GhADF7 promoter, the 5'-flanking region, including the promoter and 5'-untranslated region, was fused with the GUS gene. Histochemical assays demonstrated that the GhADF7:GUS gene was specifically expressed in pollen grains. When pollen grains germinated, very strong GUS staining was detected in the elongating pollen tube. Furthermore, overexpression of GhADF7 gene in Arabidopsis thaliana reduced the viable pollen grains and, consequently, transgenic plants were partially male-sterile. Overexpression of GhADF7 in fission yeast (Schizosaccharomyces pombe) altered the balance of actin depolymerization and polymerization, leading to the defective cytokinesis and multinucleate formation in the cells. Given all the above results together, it is proposed that the GhADF7 gene may play an important role in pollen development and germination.

Cyanide-Free Synthesis of Glycosyl Carboxylic Acids and Application for the Synthesis of Scleropentaside A.

We have developed a cyanide-free strategy for the synthesis of glycosyl carboxylic acids, which can provide 1,2-trans or 1,2-cis glycosyl carboxylic acids and is compatible with common protecting groups. The synthetic utility was demonstrated by the synthesis of 12 unreported glycosyl acids and the total synthesis of scleropentaside A.

The encapsulation of MnFe2O4 nanoparticles into the carbon framework with superior rate capability for lithium-ion batteries.

Binary transition metal oxides (BTMOs) have been regarded as one of the most hopeful anode materials for lithium-ion batteries (LIBs) owing to their high theoretical capacity, excellent electrochemical activity and abundant electrochemical reactions. However, BTMOs still suffer from two main problems, which are poor conductivity and large volume expansion during the charge/discharge processes. In order to address the above-mentioned problems, mesoporous MnFe2O4@C nanorods have been successfully synthesized in this work. The synergistic effect of the cross-linked carbon framework and mesoporous structure greatly improves the electrochemical performances. As expected, the mesoporous MnFe2O4@C electrode manifests discharge capacities of 987.5 and 816.6 mA h g-1 at the current densities of 100 and 2000 mA g-1, respectively, with the capacity retention ratio of 82.7%, exerting distinguished rate capabilities for LIBs.

A Calcium Organic Salt/rGO composite with Low Solubility and High Conductivity as a Sustainable Anode for Sodium-Ion Batteries.

Organic electrodes hold great promise for sustainable electrodes in sodium-ion batteries (SIBs) owing to their easy availability from biomass. However, traditional organic electrodes suffer from two inherent problems, high solubility in organic electrolytes and low electronic conductivity. Here, a calcium organic salt, Cabpdc (bpdc=4,4'-biphenyldicarboxylate) was designed and formed into a composite with reduced graphene oxide (rGO) to improve these two problems by a "two-in-one" approach. As expected, the Cabpdc/rGO composite displayed competitive cycle and rate performances as an anode for SIBs. Additionally, all-organic sodium-ion full cells were successfully fabricated combining this anode with a commercial organic cathode, promising applications for sustainable SIBs.

Phosphorylation dependent α-synuclein degradation monitored by in-cell NMR.

Here we report the dephosphorylation and proteolysis of phosphorylated α-synuclein, a Parkinson's disease-related protein, in living cells in a time resolved manner using in-cell NMR.