Proton Transverse Relaxation as a Sensitive Probe for Structure Determination in Solid Proteins

Solid‐state nuclear magnetic resonance (NMR) spectroscopy has been successfully applied to elucidate the atomic‐resolution structures of insoluble proteins. The major bottleneck is the difficulty to obtain valuable long‐distance structural information. Here, we propose the use of distance restraints as long as 32 Å, obtained from the quantification of transverse proton relaxation induced by a methanethiosulfonate spin label (MTSL). Combined with dipolar proton–proton distance restraints, this method allows us to obtain protein structures with excellent precision from single spin‐labeled 1 mg protein samples using fast magic angle spinning.     

Monitoring the Interaction of Albumin with a Paclitaxel-Polymer Conjugate: Complex with Warfarin as Local Probe

Summary: We have used warfarin as a local probe to investigate the orientation of paclitaxel and water soluble polymer conjugates of paxlitaxel in albumin. The relative orientation of warfarin and paclitaxel in a 1:1 complex in solution was investigated by ¹H-NMR-spektroskopy (NOESY) and the results are used for the interpretation of the steric situation of paclitaxel respectively the polymer conjugate in albumin.     

A Lysosome‐Compatible Near‐Infrared Fluorescent Probe for Targeted Monitoring of Nitric Oxide

The requirement for nitric oxide (NO) of lysosomes has motivated the development of a sophisticated fluorescent probe to monitor the distribution of this important biomolecule at the subcellular level in living cells. A near‐infrared (NIR) fluorescent Si‐rhodamine (SiRB)‐NO probe was designed based on the NO‐induced ring‐opening process of Si‐rhodamine. The probe exhibits fast chromogenic and fluorogenic responses, and high sensitivity and selectivity toward trace amounts of NO. Significantly, the spirolactam in Si‐rhodamine exhibits very good tolerance to H⁺, which in turn brings extremely low background fluorescence not only in the physiological environment but also under acidic conditions. The stability of the highly fluorescent product in acidic solution provides persistent fluorescence emission for long‐term imaging experiments. To achieve targeted imaging with improved spatial resolution and sensitivity, an efficient lysosome‐targeting moiety was conjugated to a SiRB‐NO probe, affording a tailored lysosome‐targeting NIR fluorescent Lyso‐SiRB‐NO probe. Inheriting the key advantages of its parent SiRB‐NO probe, Lyso‐SiRB‐NO is a functional probe that is suited for monitoring lysosomal NO with excellent lysosome compatibility. Imaging experiments demonstrated the monitoring of both exogenous and endogenous NO in real time by using the Lyso‐SiRB‐NO probe.     

Synthesis of SE- and TE-Containing Amino Acids: Useful Probes for Structural Studies of Proteins

ABSTRACT

The synthesis of (l)-Se-cystine, (l)-[⁷⁷Se]-cystine and (l)-Te-cystine in 4 steps from commercially available 2(S)-[(tert-butoxycarbonyl)amino]-3-hydroxy-propionate has been accomplished. [4-¹³C]Te-Met has been synthesized in a one pot reaction.     

用于监控过氧化氮的近红外荧光探针的光物理性质及PET机理

含有机硒的七甲川菁染料是基于光诱导电子转移(PET)的近红外(IR)荧光探针, 能在生理条件下高灵敏、高选择性地监控过氧化氮. 本文应用含时密度泛函理论(TD-DFT)计算方法研究其光物理性质和PET机理.结果表明, 在激发态, 荧光母体发生最高占有分子轨道(HOMO)到最低非占有分子轨道(LUMO)的电子跃迁, 识别基团上的HOMO轨道能级提高到荧光母体的单电子占据的HOMO轨道能级之上, 并向其转移一个电子, 使激发态电子回落过程受阻而导致荧光部分淬灭. 硒被氧化后, 识别基团上的HOMO轨道能级降低, PET过程被阻断, 荧光发射恢复. 研究进一步证明, PET效应来自于识别基团上苯胺N原子的p电子, 它的电子转移能力受到其对位苯硒基的氧化-还原状态的影响, 产生了荧光信号的“开-关”作用.     

Factors that Influence Competitive Intermolecular Solvation of Protonated Groups in Peptides and Proteins in the Gas Phase

The ability of 18-crown-6 (18C6) to form noncovalent complexes with cationic groups in the gas phase has been leveraged in numerous, largely orthogonal mass spectrometry-based applications. Although the fundamental interaction between 18C6 and a charged group in the gas phase is quite strong, the strength of attachment of 18C6 to large molecules is more difficult to predict because intramolecular binding of the cation can be competitive. Herein, we demonstrate in experiments with model peptides that 18C6 adducts are not strongly attached to flexible molecules with numerous potential hydrogen bonding sites. 18C6 adduct stability is increased if intramolecular charge complexation is inhibited by sterics or competitive binding. It is demonstrated with molecular mechanics that significant structural changes occur upon loss of 18C6 in model peptides. Examination of the loss of 18C6 adducts from proteins following collisional activation reveals that lower charge states lose the most 18C6. The degree of 18C6 adduct stability may reflect the degree of structural reorganization that occurs following collisional activation, suggesting that lower charge states represent structures that are not similar to gas phase idealized states. In this regard, 18C6 may serve the function of protecting solution phase protein structure. Collisional activation of holomyoglobin with 18C6 adducts attached reveals that heme loss occurs primarily after 18C6 loss, further supporting the notion that 18C6 protects native structure by solvating charged sites.

Infrared Absorption Intensity Analysis as a New Tool for Investigation of Salt Effect on Proteins

The native protein structures in buffer solution are maintained by the electrostatic force as well as the hydrophobic force, salt ions play an important role in maintaining the protein native structures, and their effect on the protein stability has attracted tremendous interests. Infrared spectroscopy has been generally used in molecular structure analysis due to its fingerprint resolution for different species including macromolecules as proteins. However spectral intensities have received much less attention than the vibrational frequencies. Here we report that the spectral intensities of protein amide I band, the finger prints for the protein secondary structures, are very sensitive to the local electric field known as Onsager reaction field caused by salt ions. IR absorbance thermal titrations have been conducted for a series of samples including simple water soluble amino acids, water soluble monomeric protein cytochrome c and dimeric protein DsbC and its single-site mutant G49R. We found that at lower temperature range （10-20℃）, there exists a thermal activated salting-in process, where the IR intensity increases with a rise in the temperature, corresponding to the ions binding of the hydrophobic surface of protein. This process is absent for the amino acids. When further raising the temperature, the IR intensity decreases, this is interpreted as the thermal activated breaking of the ion-protein surface binding. Applying Van＇t Hoff plot to the thermal titration curves, the thermodynamic parameters such as AH and AS for salting-in and ion unbinding processes can be derived for various protein secondary structural components, revealing quantitatively the extent of hydrophobic interaction as well as the strength of the ion-protein binding.     

Solvation and pairwise association in a 2D fluid

It is shown that it is possible: (a) to derive the 2D scaled particle theory formula of the reversible work of cavity creation using a geometric approach; (b) to obtain the solvation Gibbs energy in a 2D Lennard-Jones fluid; (c) to calculate the solvent contribution to the solvophobic interaction of two Lennard-Jones disks on the basis of geometric arguments. The solvent-excluded surface area associated with cavity creation decreases significantly upon pairwise association, leading to a marked increase in the configurational/translational entropy of solvent disks.     

Analytical Ultracentrifugation as a Matrix-Free Probe for the Study of Kinase Related Cellular and Bacterial Membrane Proteins and Glycans

Analytical ultracentrifugation is a versatile approach for analysing the molecular mass, molecular integrity (degradation/aggregation), oligomeric state and association/dissociation constants for self-association, and assay of ligand binding of kinase related membrane proteins and glycans. It has the great property of being matrix free—providing separation and analysis of macromolecular species without the need of a separation matrix or membrane or immobilisation onto a surface. This short review—designed for the non-hydrodynamic expert—examines the potential of modern sedimentation velocity and sedimentation equilibrium and the challenges posed for these molecules particularly those which have significant cytoplasmic or extracellular domains in addition to the transmembrane region. These different regions can generate different optimal requirements in terms of choice of the appropriate solvent (aqueous/detergent). We compare how analytical ultracentrifugation has contributed to our understanding of two kinase related cellular or bacterial protein/glycan systems (i) the membrane erythrocyte band 3 protein system—studied in aqueous and detergent based solvent systems—and (ii) what it has contributed so far to our understanding of the enterococcal VanS, the glycan ligand vancomycin and interactions of vancomycin with mucins from the gastrointestinal tract.     

A Multifunctional Chemical Probe for the Measurement of Local Micropolarity and Microviscosity in Mitochondria

The measurement of physicochemical parameters in living cells can provide information on individual cellular organelles, helping us to understand subcellular function in health and disease. While organelle‐specific chemical probes have allowed qualitative evaluation of microenvironmental variations, the simultaneous quantification of mitochondrial local microviscosity (η_m) and micropolarity (?_m), along with concurrent structural variations, has remained an unmet need. Herein, we describe a new multifunctional mitochondrial probe ( MMP ) for simultaneous monitoring of η_m and ?_m by fluorescence lifetime and emission intensity recordings, respectively. The MMP enables highly precise measurements of η_m and ?_m in the presence of a variety of agents perturbing cellular function, and the observed changes can also be correlated with alterations in mitochondrial network morphology and motility. This strategy represents a promising tool for the analysis of subtle changes in organellar structure.     

Multiple Reaction Monitoring for Direct Quantitation of Intact Proteins Using a Triple Quadrupole Mass Spectrometer

Methods that can efficiently and effectively quantify proteins are needed to support increasing demand in many bioanalytical fields. Triple quadrupole mass spectrometry (QQQ-MS) is sensitive and specific, and it is routinely used to quantify small molecules. However, low resolution fragmentation-dependent MS detection can pose inherent difficulties for intact proteins. In this research, we investigated variables that affect protein and fragment ion signals to enable protein quantitation using QQQ-MS. Collision induced dissociation gas pressure and collision energy were found to be the most crucial variables for optimization. Multiple reaction monitoring (MRM) transitions for seven standard proteins, including lysozyme, ubiquitin, cytochrome c from both equine and bovine, lactalbumin, myoglobin, and prostate-specific antigen (PSA) were determined. Assuming the eventual goal of applying such methodology is to analyze protein in biological fluids, a liquid chromatography method was developed. Calibration curves of six standard proteins (excluding PSA) were obtained to show the feasibility of intact protein quantification using QQQ-MS. Linearity (2–3 orders), limits of detection (0.5–50 μg/mL), accuracy (<5% error), and precision (1%–12% CV) were determined for each model protein. Sensitivities for different proteins varied considerably. Biological fluids, including human urine, equine plasma, and bovine plasma were used to demonstrate the specificity of the approach. The purpose of this model study was to identify, study, and demonstrate the advantages and challenges for QQQ-MS-based intact protein quantitation, a largely underutilized approach to date.

Graphical Abstract

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NMR as a Probe into the Local and Semilocal Order in Cooperative Interactions of Polyelectrolytes

Polyelectrolytes such as poly(N-diallyldimethylammonium chloride) (PDADMAC) exhibit some local and semilocal order both in their conformation dynamics (dynamic correlations) and the average density of their counterion cloud (the radial density distribution). Both aspects of order are subject to cooperative fluctuations as well as mutual reinforcement. They can be observed in particular in dilute solutions, i.e. at concentrations of the charged groups less than 5 mmol/L. At such dilution, ¹H NMR is the method of choice for the study of conformation dynamics. In addition to the recently published analysis of transverse relaxation and saturation transfer experiments, relaxations of double-quantum coherence, pseudo-solid echo and solid echo responses and exchange 2D spectra are reported here. For the radial distribution of counterions with spin-3/2 nuclei (⁷Li, ²³Na, ³⁵Cl, ⁸¹Br), the relaxation model proposed by Halle, Wennerstrom and Picullel is applied and the use of either combined longitudinal and transverse relaxation or DQC ( T ₂ ³ ) relaxation is shown. The results show that the counterion condensation, i.e. relative ordering of the counterion cloud, decreases in less densely charged polyions and vanishes for the charge separation about 2.5 nm.     

A Quadrupolar Bis-Triarylborane Chromophore as a Fluorimetric and Chirooptic Probe for Simultaneous and Selective Sensing of DNA,RNA and Proteins

A water-soluble tetracationic quadrupolar bis-triarylborane chromophore showed strong binding to ds-DNA, ds-RNA, ss-RNA, as well as to the naturally most abundant protein, BSA. The novel dye can distinguish between DNA/RNA and BSA by fluorescence emission separated by Δ =3600 cm⁻¹, allowing for the simultaneous quantification of DNA/RNA and protein (BSA) in a mixture. The applicability of such fluorimetric differentiation in vitro was demonstrated, strongly supporting a protein-like target as a dominant binding site of 1 in cells. Moreover, our dye also bound strongly to ss-RNA, with the unusual rod-like structure of the dye, decorated by four positive charges at its termini and having a hydrophobic core, acting as a spindle for wrapping A, C and U ss-RNAs, but not poly G, the latter preserving its secondary structure. To the best of our knowledge, such unmatched, multifaceted binding activity of a small molecule toward DNA, RNA, and proteins and the selectivity of its fluorimetric and chirooptic response makes the quadrupolar bis-triarylborane a novel chromophore/fluorophore moiety for biochemical applications.     

基于扫描探针显微术的微区光电性质测试方法和装置

本文以一台常规的Aglient 5500型的扫描探针显微镜为基础,通过配置光源及聚焦系统、研制光强度自动控制部件、研制样品台等方法将太阳光模拟器发射的光引入样品的探针扫描区域,开发了利用扫描探针显微镜原位测试太阳能电池材料微区光电性质的功能。光电性质的测试过程可在手套箱内进行,解决了有机半导体容易吸收空气中的水和氧而失效的问题。     

Development of a DNA‐Templated Peptide Probe for Photoaffinity Labeling and Enrichment of the Histone Modification Reader Proteins

Histone post‐translational modifications (HPTMs) provide signal platforms to recruit proteins or protein complexes to regulate gene expression. Therefore, the identification of these recruited partners (readers) is essential to understand the underlying regulatory mechanisms. However, it is still a major challenge to profile these partners because their interactions with HPTMs are rather weak and highly dynamic. Herein we report the development of a HPTM dual probe based on DNA‐templated technology and a photo‐crosslinking method for the identification of HPTM readers. By using the trimethylation of histone H3 lysine 4, we demonstrated that this HPTM dual probe can be successfully utilized for labeling and enrichment of HPTM readers, as well as for the discovery of potential HPTM partners. This study describes the development of a new chemical proteomics tool for profiling HPTM readers and can be adapted for broad biomedical applications.     

A Selective Near‐Infrared Fluorescent Probe for In Vivo Imaging of Thiophenols from a Focused Library

Thiophenols are highly toxic industrial materials that, once released, will accumulate in the environment, and ultimately in human bodies, thereby causing serious health problems. To achieve their selective and sensitive detection, a novel near‐infrared (NIR) fluorescent probe ( CCP‐1 ) from a focused library was developed for thiophenol species. Our studies show that CCP‐1 displays a thiophenol‐triggered 28‐fold fluorescence intensity enhancement at 706 nm, with a detection limit of 34 nm observed. It is also able to differentiate thiophenols from various other thiol‐containing analytes including hydrogen sulfide, hydrogen persulfide, and aliphatic thiols. In total, the desirable properties (e.g., excitation/emission in the NIR region, good cell‐membrane permeability, intracellular stability, and low cytotoxicity) make CCP‐1 a potential candidate for thiophenol detection both in vitro and in vivo. In addition, CCP‐1 , for the first time, successfully visualized thiophenols in mice models of thiophenol inhalation.     

Construction of a Near‐Infrared Fluorescent Turn‐On Probe for Selenol and Its Bioimaging Application in Living Animals

As selenocysteine (Sec) carries out the majority of the functions of the various Se‐containing species in vivo, it is of high importance to develop reliable and rapid assays with biocompatibility to detect Sec. Herein, an NIR fluorescent turn‐on probe for highly selective detection of selenol was designed and synthesized. The probe exhibits large turn‐on signal upon treatment with selenocysteine (R‐SeH), and it was further demonstrated that the new NIR fluorescent probe can be employed to image selenol in living animals.