C-terminal domain dimerization in yeast Hsp90 is moderately modulated by the other domains.

Heat shock protein 90 (Hsp90) serves as a crucial regulator of cellular proteostasis by stabilizing and regulating the activity of numerous substrates, many of which are oncogenic proteins. Therefore, Hsp90 is a drug target for cancer therapy. Hsp90 comprises three structural domains, a highly conserved amino-terminal domain (NTD), a middle domain (MD), and a carboxyl-terminal domain (CTD). The CTD is responsible for protein dimerization, is crucial for Hsp90's activity, and has therefore been targeted for inhibiting Hsp90. Here we addressed the question of whether the CTD dimerization in Hsp90, in the absence of bound nucleotides, is modulated by allosteric effects from the other domains. We studied full length (FL) and isolated CTD (isoC) yeast Hsp90 spin-labeled with a Gd(III) tag by double electron-electron resonance measurements to track structural differences and to determine the apparent dissociation constant (Kd). We found the distance distributions for both the FL and isoC to be similar, indicating that the removal of the NTD and MD does not significantly affect the structure of the CTD dimer. The low-temperature double electron-electron resonance-derived Kd values, as well as those obtained at room temperature using microscale thermophoresis and native mass spectrometry, collectively suggested the presence of some allosteric effects from the NTDs and MDs on the CTD dimerization stability in the apo state. This was evidenced by a moderate increase in the Kd for the isoC compared with the FL mutants. Our results reveal a fine regulation of the CTD dimerization by allosteric modulation, which may have implications for drug targeting strategies in cancer therapy.

Genetically Encoded Photocaged Proteinogenic and Non-Proteinogenic Amino Acids.

Photocaged amino acids could be genetically encoded into proteins via genetic code expansion (GCE) and constitute unique tools for innovative protein engineering. There are a number of photocaged proteinogenic amino acids that allow strategic conversion of proteins into their photocaged variants, thus enabling spatiotemporal and non-invasive regulation of protein functions using light. Meanwhile, there are a hand of photocaged non-proteinogenic amino acids that address the challenges in directly encoding certain non-canonical amino acids (ncAAs) that structurally resemble proteinogenic ones or possess highly reactive functional groups. Herein, we would like to summarize the efforts in encoding photocaged proteinogenic and non-proteinogenic amino acids, hoping to draw more attention to this fruitful and exciting scientific campaign.

Site-Specific Anchoring a Luminescent Tag in a Protein with Non-Emissive Iridium(III) Complex.

Site-specific modification of proteins with synthetic fluorescent tag effectively improves the resolution of imaging, and such a labeling method with negligible three-dimensional structural perturbations and minimal impact on the biological functions of proteins is of high interest to dissect the high-resolution activities of biomolecules in complex systems. To this end, several non-emissive iridium(III) complexes [Ir(C-N)2 (H2 O)2 ]+ OTF- (C-N denotes various cyclometalated ligands) were designed and synthesized. These complexes were tested for attaching a protein by coordinating to H/X (HisMet, HisHis, and HisCys) that are separated by i and i+4 in α-helix. Replacement of the two labile water ligands in the iridium(III) complex by a protein HisHis pair increases the luminescent intensity up to over 100 folds. This labeling approach has been demonstrated in a highly specific and efficient manner in a number of proteins, and it is also feasible for labeling target proteins in cell lysates.

Critical role of minor eggcase silk component in promoting spidroin chain alignment and strong fiber formation.

Natural spider silk with extraordinary mechanical properties is typically spun from more than one type of spidroin. Although the main components of various spider silks have been widely studied, little is known about the molecular role of the minor silk components in spidroin self-assembly and fiber formation. Here, we show that the minor component of spider eggcase silk, TuSp2, not only accelerates self-assembly but remarkably promotes molecular chain alignment of spidroins upon physical shearing. NMR structure of the repetitive domain of TuSp2 reveals that its dimeric structure with unique charged surface serves as a platform to recruit different domains of the main eggcase component TuSp1. Artificial fiber spun from the complex between TuSp1 and TuSp2 minispidroins exhibits considerably higher strength and Young's modulus than its native counterpart. These results create a framework for rationally designing silk biomaterials based on distinct roles of silk components.

Elevated levels of Zn, Cu and Co are associated with an increased risk of endometriosis: Results from a casecontrol study.

BACKGROUND: Endometriosis is a common gynecological disease that affects approximately 5 %∼10 % of reproductive-aged women. Zinc (Zn), selenium (Se), copper (Cu), cobalt (Co) and molybdenum (Mo) are essential trace elements and are very important for human health. However, studies on the relationship between mixtures of essential trace elements and the risk of endometriosis are limited and inconsistent. In particular, studies confirming the association via different sample types are limited. OBJECTIVE: This study aimed to investigate the associations between Zn, Se, Cu, Co and Mo concentrations in blood and follicular fluid (FF) and endometriosis risk in a Chinese population. METHODS: A total of 609 subjects undergoing in vitro fertilization (IVF) were recruited; 836 samples were analyzed, including 451 blood samples (234 controls and 217 cases) and 385 FF samples (203 controls and 182 cases). In addition, 227 subjects provided both blood and FF samples. Zn, Se, Cu, Co and Mo concentrations in blood and FF were quantified via inductively coupled plasma-mass spectrometry (ICP-MS). The associations between the levels of Zn, Se, Cu, Co and Mo and the risk of endometriosis were assessed using single-element models (logistic regression models), and the combined effect of the trace elements on endometriosis risk was assessed using multielement models (Bayesian kernel machine regression (BKMR) and weighted quantile sum (WQS) regression). RESULTS: Based on the single-element models, significant associations of Zn concentrations in blood (high-level vs. low-level group: aOR = 14.17, 95 % CI: 7.31, 27.50) and FF (first tertile vs. second tertile group: aOR = 0.34, 95 % CI: 0.16, 0.71; third tertile vs. second tertile group: aOR = 2.32, 95 % CI: 1.38, 3.91, respectively) and Co concentrations in blood (first tertile vs. second tertile group, aOR = 0.24, 95 % CI: 0.12, 0.48) and FF (third tertile vs. second tertile group: aOR = 3.87, 95 % CI: 2.19, 6.84) with endometriosis risk were found after adjustment for all confounders. In FF, Cu and Mo levels were significantly greater among the cases than among the controls, with a positive association with endometriosis risk (Cu (first tertile vs. second tertile group: aOR = 0.39, 95 % CI: 0.19, 0.81; third tertile vs. second tertile group: aOR = 2.73, 95 % CI: 1.61, 4.66, respectively) and Mo (high-level vs. low-level group: aOR = 14.93, 95 % CI: 7.16, 31.12)). However, similar associations between blood Cu and Mo levels and endometriosis risk were not found. In addition, the levels of these five essential trace element mixtures in blood and in FF were significantly and positively associated with endometriosis risk according to the BKMR analyses; the levels of Zn and Cu in blood and the levels of Mo in FF were significantly related to the risk of endometriosis, and the posterior inclusion probabilities (PIPs) were 1.00, 0.99 and 1.00 for Zn and Cu levels in blood and Mo levels in FF, respectively. Furthermore, Zn and Mo were the highest weighted elements in blood and FF, respectively, according to WQS analyses. CONCLUSION: The risk of endometriosis was associated with elevated levels of several essential trace elements (Zn, Cu and Co). Elevated levels of these elements may be involved in the pathomechanism of endometriosis. However, further studies with larger sample sizes will be necessary to confirm these associations.

Finite Element Modeling and Calibration of a Three-Span Continuous Suspension Bridge Based on Loop Adjustment and Temperature Correction.

Precise finite element modeling is critically important for the construction and maintenance of long-span suspension bridges. During the process of modeling, shape-finding and model calibration directly impact the accuracy and reliability. Scholars have provided numerous alternative proposals for the shape-finding of main cables in suspension bridges from both theoretical and finite element analysis perspectives. However, it is difficult to apply these solutions to suspension bridges with special components. Seeking a viable solution for such suspension bridges holds practical significance. The Nanjing Qixiashan Yangtze River Bridge is the first three-span suspension bridge in China. To maintain the configuration of the main cable, the suspension bridge is equipped with specialized suspenders near the anchors, referred to as displacement-limiting suspenders. It is the first suspension bridge in China to use displacement-limiting suspenders and their anchorage system. Taking the suspension bridge as a research background, this paper introduces a refined finite element modeling approach considering the effect of geometric nonlinearity. Firstly, based on the loop adjustment and temperature correction, the shape-finding and force assessment of the main cables are carried out. On this basis, a nonlinear finite element model of the bridge was established and calibrated, taking into account factors such as pylon settlement and cable saddle precession. Finally, the static and dynamic characteristics of the suspension bridge were thoroughly investigated. This study aims to provide a reference for the design, construction and operation of the three-span continuous suspension bridge.

Relaxation-Assisted Magnetization Transfer Phenomena for a Sensitivity-Enhanced 2D NMR.

2D NOESY and TOCSY play central roles in contemporary NMR. We have recently discussed how solvent-driven exchanges can significantly enhance the sensitivity of such methods when attempting correlations between labile and nonlabile protons. This study explores two scenarios where similar sensitivity enhancements can be achieved in the absence of solvent exchange: the first one involves biomolecular paramagnetic systems, while the other involves small organic molecules in natural abundance. It is shown that, in both cases, the effects introduced by either differential paramagnetic shift and relaxation or by polarization sharing among networks of protons can provide a similar sensitivity boost, as previously discussed for solvent exchange. The origin and potential of the resulting enhancements are analyzed, and experiments that demonstrate them in protein and natural products are exemplified. Limitations and future improvements of these approaches are also briefly discussed.

Stabilizing Nitroxide Spin Labels for Structural and Conformational Studies of Biomolecules by Maleimide Treatment.

Nitroxide (NO) spin radicals are effective in characterizing structures, interactions and dynamics of biomolecules. The EPR applications in cell lysates or intracellular milieu require stable spin labels, but NO radicals are unstable in such conditions. We showed that the destabilization of NO radicals in cell lysates or even in cells is caused by NADPH/NADH related enzymes, but not by the commonly believed reducing reagents such as GSH. Maleimide stabilizes the NO radicals in the cell lysates by consumption of the NADPH/NADH that are essential for the enzymes involved in destabilizing NO radicals, instead of serving as the solo thiol scavenger. The maleimide treatment retains the crowding properties of the intracellular components and allows to perform long-time EPR measurements of NO labeled biomolecules close to the intracellular conditions. The strategy of maleimide treatment on cell lysates for the EPR applications has been demonstrated on double electron-electron resonance (DEER) measurements on a number of NO labeled protein samples. The method opens a broad application range for the NO labeled biomolecules by EPR in conditions that resemble the intracellular milieu.

Stereospecific recognition of a chiral centre over multiple flexible covalent bonds by 19F-NMR.

High performance in chiral recognition by a reactive 19F-tag was demonstrated for a variety of enantiomers. The analytes with up to five flexible covalent bonds from the chiral center can be discriminated by a sensitive chiral reporter manifested in the 19F-NMR spectrum. Simultaneous identification of chiral amines in a mixture and high accuracy ee determination were achieved.

In-cell destabilization of a homodimeric protein complex detected by DEER spectroscopy.

The complexity of the cellular medium can affect proteins' properties, and, therefore, in-cell characterization of proteins is essential. We explored the stability and conformation of the first baculoviral IAP repeat (BIR) domain of X chromosome-linked inhibitor of apoptosis (XIAP), BIR1, as a model for a homodimer protein in human HeLa cells. We employed double electron-electron resonance (DEER) spectroscopy and labeling with redox stable and rigid Gd3+ spin labels at three representative protein residues, C12 (flexible region), E22C, and N28C (part of helical residues 26 to 31) in the N-terminal region. In contrast to predictions by excluded-volume crowding theory, the dimer-monomer dissociation constant KD was markedly higher in cells than in solution and dilute cell lysate. As expected, this increase was partially recapitulated under conditions of high salt concentrations, given that conserved salt bridges at the dimer interface are critically required for association. Unexpectedly, however, also the addition of the crowding agent Ficoll destabilized the dimer while the addition of bovine serum albumin (BSA) and lysozyme, often used to represent interaction with charged macromolecules, had no effect. Our results highlight the potential of DEER for in-cell study of proteins as well as the complexities of the effects of the cellular milieu on protein structures and stability.

Two closed ATP- and ADP-dependent conformations in yeast Hsp90 chaperone detected by Mn(II) EPR spectroscopic techniques.

Hsp90 plays a central role in cell homeostasis by assisting folding and maturation of a large variety of clients. It is a homo-dimer, which functions via hydrolysis of ATP-coupled to conformational changes. Hsp90's conformational cycle in the absence of cochaperones is currently postulated as apo-Hsp90 being an ensemble of "open"/"closed" conformations. Upon ATP binding, Hsp90 adopts an active ATP-bound closed conformation where the N-terminal domains, which comprise the ATP binding site, are in close contact. However, there is no consensus regarding the conformation of the ADP-bound Hsp90, which is considered important for client release. In this work, we tracked the conformational states of yeast Hsp90 at various stages of ATP hydrolysis in frozen solutions employing electron paramagnetic resonance (EPR) techniques, particularly double electron-electron resonance (DEER) distance measurements. Using rigid Gd(III) spin labels, we found the C domains to be dimerized with same distance distribution at all hydrolysis states. Then, we substituted the ATPase Mg(II) cofactor with paramagnetic Mn(II) and followed the hydrolysis state using hyperfine spectroscopy and measured the inter-N-domain distance distributions via Mn(II)-Mn(II) DEER. The point character of the Mn(II) spin label allowed us resolve 2 different closed states: The ATP-bound (prehydrolysis) characterized by a distance distribution having a maximum of 4.3 nm, which broadened and shortened, shifting the mean to 3.8 nm at the ADP-bound state (posthydrolysis). This provides experimental evidence to a second closed conformational state of Hsp90 in solution, referred to as "compact." Finally, the so-called high-energy state, trapped by addition of vanadate, was found structurally similar to the posthydrolysis state.

Novel economical, accurate, sensitive, single-cell analytical method for mitochondrial DNA quantification in mtDNA mutation carriers.

PURPOSE: Although a variety of analytical methods have been developed to detect mitochondrial DNA (mtDNA) heteroplasmy, there are special requirements of mtDNA heteroplasmy quantification for women carrying mtDNA mutations receiving the preimplantation genetic diagnosis (PGD) and prenatal diagnosis (PD) in clinic. These special requirements include various sample types, large sample number, long-term follow-up, and the need for detection of single-cell from biopsied embryos. Therefore, developing an economical, accurate, high-sensitive, and single-cell analytical method for mtDNA heteroplasmy is necessary. METHODS: In this study, we developed the Sanger sequencing combined droplet digital polymerase chain reaction (ddPCR) method for mtDNA quantification and compared the results to next-generation sequencing (NGS). A total of seventeen families with twelve mtDNA mutations were recruited in this study. RESULTS: The results showed that both Sanger sequencing and ddPCR could be used to analyze the mtDNA heteroplasmy in single-cell samples. There was no statistically significant difference in heteroplasmy levels in common samples with high heteroplasmy (≥ 5%), low heteroplasmy (< 5%), and single-cell samples, either between Sanger sequencing and NGS methods, or between ddPCR and NGS methods (P > 0.05). However, Sanger sequencing was unable to detect extremely low heteroplasmy accurately. But even in samples with extremely low heteroplasmy (0.40% and 0.92%), ddPCR was always able to quantify them. Compared to NGS, Sanger sequencing combined ddPCR analytical methods greatly reduced the cost of sequencing. CONCLUSIONS: In conclusion, this study successfully established an economical, accurate, sensitive, single-cell analytical method based on the Sanger sequencing combined ddPCR methods for mtDNA heteroplasmy quantification in a clinical setting.

A phenomenographic study of Chinese primary school students' conceptions about technology.

Although there are some researches conducted about students' conceptions of technology, little research has been conducted to reveal the primary school students' conceptions concerning technology in China. This research investigated Chinese primary school students' (aged 9-12) conceptions of technology as regards their understanding of (a) the concept of technology, (b) the impact of technology on human life and nature, and (c) the relationship between technology and science. Phenomenography as the methodological framework was adopted for this study. A total of 63 primary school students were chosen as participants in the study to probe their conceptions about technology through picture/photo eliciting activities, and semi-structured, personal interviews in a website video format. It is found that the primary school students defined technology from diverse perspectives, including the dimensions of its attributes, production, operation and use, function, with most of them regarding technology as a double-edged sword. It is also found that they lack a comprehensive and rational understanding of the concept of technology and cannot understand the relationship between science and technology properly. This study contributes better to understanding primary school students' conceptions about technology in mainland China and beyond, thus providing an empirical basis for improving technology education policy, curriculum, instruction, and assessment in the future for China and other countries.

Constructing Photoactivatable Protein with Genetically Encoded Photocaged Glutamic Acid.

Genetically replacing an essential residue with the corresponding photocaged analogues via genetic code expansion (GCE) constitutes a useful and unique strategy to directly and effectively generate photoactivatable proteins. However, the application of this strategy is severely hampered by the limited number of encoded photocaged proteinogenic amino acids. Herein, we report the genetic incorporation of photocaged glutamic acid analogues in E. coli and mammalian cells and demonstrate their use in constructing photoactivatable variants of various fluorescent proteins and SpyCatcher. We believe genetically encoded photocaged Glu would significantly promote the design and application of photoactivatable proteins in many areas.

GdIII -19 F Distance Measurements for Proteins in Cells by Electron-Nuclear Double Resonance.

Studies of protein structure and dynamics are usually carried out in dilute buffer solutions, conditions that differ significantly from the crowded environment in the cell. The double electron-electron resonance (DEER) technique can track proteins' conformations in the cell by providing distance distributions between two attached spin labels. This technique, however, cannot access distances below 1.8 nm. Here, we show that GdIII -19 F Mims electron-nuclear double resonance (ENDOR) measurements can cover part of this short range. Low temperature solution and in-cell ENDOR measurements, complemented with room temperature solution and in-cell GdIII -19 F PRE (paramagnetic relaxation enhancement) NMR measurements, were performed on fluorinated GB1 and ubiquitin (Ub), spin-labeled with rigid GdIII tags. The proteins were delivered into human cells via electroporation. The solution and in-cell derived GdIII -19 F distances were essentially identical and lie in the 1-1.5 nm range revealing that both, GB1 and Ub, retained their overall structure in the GdIII and 19 F regions in the cell.

Distinct stereospecific effect of chiral tether between a tag and protein on the rigidity of paramagnetic tag.

Flexibility between the paramagnetic tag and its protein conjugates is a common yet unresolved issue in the applications of paramagnetic NMR spectroscopy in biological systems. The flexibility greatly attenuates the magnetic anisotropy and compromises paramagnetic effects especially for pseudocontact shift and residual dipolar couplings. Great efforts have been made to improve the rigidity of paramagnetic tag in the protein conjugates, however, the effect of local environment vicinal to the protein ligation site on the paramagnetic effects remains poorly understood. In the present work, the stereospecific effect of chiral tether between the protein and a tag on the paramagnetic effects produced by the tag attached via a D- and L-type linker between the protein and paramagnetic metal chelating moiety was assessed. The remarkable chiral effect of the D- and L-type tether between the tag and the protein on the rigidity of paramagnetic tag is disclosed in a number of protein-tag-Ln complexes. The chiral tether formed between the D-type tag and L-type protein surface minimizes the effect of the local environment surrounding the ligation site on the averaging of paramagnetic tag, which is helpful to preserve the rigidity of a paramagnetic tag in the protein conjugates.

Simultaneous Discrimination and Quantification of Enantiomeric Amino Acids under Physiological Conditions by Chiral 19F NMR Tag.

Enantiomeric analysis is of great significance in chemistry, chemical biology and pharmaceutical research. We herein propose a chiral 19F NMR tag containing an amino reactive NHS group to discriminate the enantiomeric amino acids under physiological conditions by NMR spectroscopy. The chiral 19F NMR tag readily forms stable amide products with the amino acids in aqueous solution. The stereospecific chemistry of enantiomeric amino acids is discriminated by a stereogenic carbon bonded with a 19F atom and is therefore decoded by the 19F reporter and manifested in the distinct 19F chemical shift. The chemical shift difference (ΔΔδ) of the chiral 19F NMR tag derived enantiomeric amino acids variants has a broad chemical shift range between -1.13 to 1.68 ppm, indicating the high sensitivity of the chiral 19F NMR tag to the stereospecific environment surrounding the individual amino acids. The distinguishable chemical shift produced by the chiral 19F NMR tag permits simultaneous discrimination and quantification of the enantiomeric amino acids in a mixture. The high fidelity of the chiral 19F NMR tag affords high-accuracy determination of the enantiomeric composition of amino acids by simple 1D NMR under physiological conditions.

Simultaneous Quantification of Biothiols and Deciphering Diverse GSH Stability in Different Live Cells by 19F-Tag.

GSH, Cys, Hcy, and H2S are important biothiols and play important roles in the living systems. Quantitative and simultaneous determination of these biothiols under physiological conditions is still a challenge. Herein, we developed an effective 19F-reactive tag that readily interacts with these four biothiols for the generation of stable thioether products that have distinguishable 19F-chemical shifts. These thioester compounds encode the characteristic fingerprint profiles of each biothiols, allowing one to simultaneously quantify and determine these biothiols by 1D 19F NMR spectroscopy. The intra-/extracellular GSH in live cells was assessed by the established strategy, and remarkable variations in the GSH stability were determined between the normal mammalian cells and cancer cells. It is notable that GSH hydrolyzes efficiently in the out-membrane of the cancer cells and the lysates. In contrast, GSH remains stable in the tested normal cells.

Visualizing Proteins in Mammalian Cells by 19 F NMR Spectroscopy.

In-cell NMR spectroscopy is a powerful tool to investigate protein behavior in physiologically relevant environments. Although proven valuable for disordered proteins, we show that in commonly used 1 H-15 N HSQC spectra of globular proteins, interactions with cellular components often broaden resonances beyond detection. This contrasts 19 F spectra in mammalian cells, in which signals are readily observed. Using several proteins, we demonstrate that surface charges and interaction with cellular binding partners modulate linewidths and resonance frequencies. Importantly, we establish that 19 F paramagnetic relaxation enhancements using stable, rigid Ln(III) chelate pendants, attached via non-reducible thioether bonds, provide an effective means to obtain accurate distances for assessing protein conformations in the cellular milieu.

Efficient Protein-Protein Couplings Mediated by Small Molecules under Mild Conditions.

Protein-protein coupling reactions under physiological conditions that do not impact the three-dimensional structures of the proteins are in high demand. Owing to the combination of phenylsulfonyl and aldehyde groups in 5-fluoro-4-(phenylsulfonyl)picolinaldehyde (FPPA), the fluorine substituent shows high reactivity toward free thiols. In FPPA, the fluorine is more reactive than phenylsulfonyl for free thiols. Thus the first quantitative nucleophilic substitution can be followed by selective substitution of phenylsulfonyl by an additional thiol or cyclization of aldehyde with a 1,2-aminothiol molecule. The FPPA mediated protein-protein coupling proceeds efficiently under mild conditions, resulting in stable protein conjugates. This coupling method has negligible 3D structural perturbations on the target proteins, and it produces overall intact, nearly traceless, and native structural folds of proteins. It is highly suitable for reconstruction of proteins that are difficult to make and segmental isotopic labeling of multidomain proteins.