Simultaneous measurement of N?H and Cα?Hα coupling constants in proteins

We present a pulse sequence for the simultaneous measurement of N? H and Cα? Hα couplings in double‐labeled proteins from 2D spectra. The proposed sequence, a modification of the HN(CO)CA experiment, combines the J‐modulation method and the IPAP scheme. The couplings can be readily retrieved from a series of 2D ¹⁵N? ¹H correlation spectra, differing in the time point at which a ¹H 180° pulse is applied. This induces an intensity modulation of the ¹⁵N? ¹H correlation peaks with the Cα? Hα coupling. The Cα? Hα coupling is then obtained by fitting the observed intensities to the modulation equation. The N? H coupling is measured in each member of the set from peak‐to‐peak separations in the IPAP subspectra. The pulse sequence is experimentally verified with a sample of ¹⁵N/¹³C‐enriched ubiquitin. Copyright © 2009 John Wiley & Sons, Ltd.     

Ultrafast Vibrational Dynamics of Membrane‐Bound Peptides at the Lipid Bilayer/Water Interface

Vibrational energy transfer (VET) of proteins at cell membrane plays critical roles in controlling the protein functionalities, but its detection is very challenging. By using a surface‐sensitive femtosecond time‐resolved sum‐frequency generation vibrational spectroscopy with infrared pump, the detection of the ultrafast VET in proteins at cell membrane has finally become possible. The vibrational relaxation time of the N−H groups is determined to be 1.70(±0.05) ps for the α‐helix located in the hydrophobic core of the lipid bilayer and 0.9(±0.05) ps for the membrane‐bound β‐sheet structure. The N−H groups with strong hydrogen bonding gain faster relaxation time. By pumping the amide A band and probing amide I band, the vibrational relaxation from N−H mode to C=O mode through two pathways (direct coupling and through intermediate states) is revealed. The ratio of the pathways depends on the NH⋅⋅⋅O=C hydrogen‐bonding strength. Strong hydrogen bonding favors the coupling through intermediate states.     

An Efficient Labelling Approach to Harness Backbone and Side‐Chain Protons in 1H‐Detected Solid‐State NMR Spectroscopy

¹H‐detection can greatly improve spectral sensitivity in biological solid‐state NMR (ssNMR), thus allowing the study of larger and more complex proteins. However, the general requirement to perdeuterate proteins critically curtails the potential of ¹H‐detection by the loss of aliphatic side‐chain protons, which are important probes for protein structure and function. Introduced herein is a labelling scheme for ¹H‐detected ssNMR, and it gives high quality spectra for both side‐chain and backbone protons, and allows quantitative assignments and aids in probing interresidual contacts. Excellent ¹H resolution in membrane proteins is obtained, the topology and dynamics of an ion channel were studied. This labelling scheme will open new avenues for the study of challenging proteins by ssNMR.     

PCASSO: A fast and efficient Cα‐based method for accurately assigning protein secondary structure elements

Proteins are often characterized in terms of their primary, secondary, tertiary, and quaternary structure. Algorithms such as define secondary structure of proteins (DSSP) can automatically assign protein secondary structure based on the backbone hydrogen‐bonding pattern. However, the assignment of secondary structure elements (SSEs) becomes a challenge when only the Cα coordinates are available. In this work, we present protein C‐alpha secondary structure output (PCASSO), a fast and accurate program for assigning protein SSEs using only the Cα positions. PCASSO achieves ～95% accuracy with respect to DSSP and takes ～0.1 s using a single processor to analyze a 1000 residue system with multiple chains. Our approach was compared with current state‐of‐the‐art Cα‐based methods and was found to outperform all of them in both speed and accuracy. A practical application is also presented and discussed. © 2014 Wiley Periodicals, Inc.     

1H‐Detected Solid‐State NMR Studies of Water‐Inaccessible Proteins In Vitro and In Situ

¹H detection can significantly improve solid‐state NMR spectral sensitivity and thereby allows studying more complex proteins. However, the common prerequisite for ¹H detection is the introduction of exchangeable protons in otherwise deuterated proteins, which has thus far significantly hampered studies of partly water‐inaccessible proteins, such as membrane proteins. Herein, we present an approach that enables high‐resolution ¹H‐detected solid‐state NMR (ssNMR) studies of water‐inaccessible proteins, and that even works in highly complex environments such as cellular surfaces. In particular, the method was applied to study the K⁺ channel KcsA in liposomes and in situ in native bacterial cell membranes. We used our data for a dynamic analysis, and we show that the selectivity filter, which is responsible for ion conduction and highly conserved in K⁺ channels, undergoes pronounced molecular motion. We expect this approach to open new avenues for biomolecular ssNMR.     

An Approach to NMR Assignment of Intrinsically Disordered Proteins

An efficient approach to NMR assignments in intrinsically disordered proteins is presented, making use of the good dispersion of cross peaks observed in [¹⁵N,¹³C′]‐ and [¹³C′,¹H^N]‐correlation spectra. The method involves the simultaneous collection of {3D (H)NCO(CAN)H and 3D (HACA)CON(CA)HA} spectra for backbone assignments via sequential H^N and H^α correlations and {3D (H)NCO(CACS)HS and 3D (HS)CS(CA)CO(N)H} spectra for side‐chain ¹H and ¹³C assignments, employing sequential ¹H data acquisitions with direct detection of both the amide and aliphatic protons. The efficacy of the approach for obtaining resonance assignments with complete backbone and side‐chain chemical shifts is demonstrated experimentally for the 61‐residue [¹³C,¹⁵N]‐labelled peptide of a voltage‐gated potassium channel protein of the Kv1.4 channel subunit. The general applicability of the approach for the characterisation of moderately sized globular proteins is also demonstrated.     

Systematical NMR analysis of swainsonine,a mycotoxin from endophytic fungus Alternaria oxytropis

Swainsonine (SW, 1 ), a unique indolizine with poly‐hydroxyl groups, was re‐isolated from the plant endophytic fungus Alternaria oxytropis. The structure (including planar structure and relative configuration) was systematically elucidated by NMR spectra (including ¹H, ¹³C, ¹H‐¹H COSY, HMQC, HMBC, and NOESY spectra in DMSO‐d₆ and in CD₃OD); ¹H NMR spectra of the modified Mosher's products were first used to determine the absolute configuration of SW. More importantly, the complex coupled features of H‐7α, H‐7β, and H‐6α in the ¹H NMR spectrum of ( 1 ) were analyzed in details, which will provide aids for the planar and relative configuration determination of analogs.     

Selective lighting up of segments around Gly,Ala and Ser/Thr in proteins

Direct detection of ¹³C nucleus can be used as a valuable alternative where ¹H detection poses a challenge due to relaxation effects, chemical exchange and poor chemical shift dispersion. In this context, we have designed a suite of 2D ¹³C^α‐detected hNCA experiments that provide sequential correlations of ¹³C^α with ¹⁵N on one hand and efficient spectroscopic labeling of certain groups of residues, namely, Gly, Ala, Ser and Thr, on the other. These residues act as checkpoints in the sequential walk, which in turn offer new possibilities of backbone assignment of small proteins from a set of 2D experiments, thereby providing great economy in terms of spectrometer time. The direct identification of peptide segments around Gly, Ala, Ser and Thr residues along a protein chain will be highly valuable for deriving important information on sites of ligand binding, phosphorylation, inhibitor/substrate binding, understanding protein folding pathways, comprehending local conformational dynamics etc. without having to obtain complete sequence‐specific assignments, which can be time consuming and at times formidable, especially in large proteins. We have illustratively demonstrated the multifaceted applications of these variants of 2D experiments on ubiquitin and M‐crystallin. We foresee that these 2D hNCA experiments will provide economic and efficient strategies for studying the structure and function of proteins. Copyright © 2012 John Wiley & Sons, Ltd.     

Observation of CH⋅⋅⋅π Interactions between Methyl and Carbonyl Groups in Proteins

Protein structure and function is dependent on myriad noncovalent interactions. Direct detection and characterization of these weak interactions in large biomolecules, such as proteins, is experimentally challenging. Herein, we report the first observation and measurement of long‐range “through‐space” scalar couplings between methyl and backbone carbonyl groups in proteins. These J couplings are indicative of the presence of noncovalent C−H⋅⋅⋅π hydrogen‐bond‐like interactions involving the amide π network. Experimentally detected scalar couplings were corroborated by a natural bond orbital analysis, which revealed the orbital nature of the interaction and the origins of the through‐space J couplings. The experimental observation of this type of CH⋅⋅⋅π interaction adds a new dimension to the study of protein structure, function, and dynamics by NMR spectroscopy.     

13C direct detection experiments on the paramagnetic oxidized monomeric copper, zinc superoxide dismutase

In this report, the use of 13C direct detection has been pursued in 2D experiments (13C-13C COSY, 13C-13C COCAMQ, 13C-13C NOESY) to detect broad lines in nuclear magnetic resonance spectra of paramagnetic metalloproteins. The sample is a monomeric oxidized copper, zinc superoxide dismutase. Thanks to direct detection probeheads, cryogenic technology, and implementation of 13C band-selective homodecoupling, many broadened signals were detected. Proton signals for the same residues escaped detection in 1H and 1H-15N HSQC experiments because of the broadening. Only the 13C signals which experience large contact coupling escaped detection, i.e., the 13C nuclei of the metal coordinated histidines. Otherwise, nuclei as close to copper(II) as 4 A can be detected. Paramagnetic-based restraints can in principle be used for solution structure determination of paramagnetic metalloproteins and in copper(II) proteins in particular. The present study is significant also for the study of large diamagnetic proteins for which proton relaxation makes proton-based spectroscopy not adequate.     

Quadruple‐Resonance Magic‐Angle Spinning NMR Spectroscopy of Deuterated Solid Proteins

¹H‐detected magic‐angle spinning NMR experiments facilitate structural biology of solid proteins, which requires using deuterated proteins. However, often amide protons cannot be back‐exchanged sufficiently, because of a possible lack of solvent exposure. For such systems, using ²H excitation instead of ¹H excitation can be beneficial because of the larger abundance and shorter longitudinal relaxation time, T₁, of deuterium. A new structure determination approach, “quadruple‐resonance NMR spectroscopy”, is presented which relies on an efficient ²H‐excitation and ²H‐¹³C cross‐polarization (CP) step, combined with ¹H detection. We show that by using ²H‐excited experiments better sensitivity is possible on an SH3 sample recrystallized from 30 % H₂O. For a membrane protein, the ABC transporter ArtMP in native lipid bilayers, different sets of signals can be observed from different initial polarization pathways, which can be evaluated further to extract structural properties.     

Piezoelectric Quartz Crystal Anti‐Protein Immunosensors Based on Immobilized Fullerene C60‐Proteins

Various fullerene C60‐proteins such as C60‐myoglobin (C60‐Mb), C60‐hemoglobin (C60‐Hb) and C60‐gliadin, coated piezoelectric quartz crystals were prepared and applied in piezoelectric quartz crystal immunosensors for protein‐antibodies such as anti‐myoglobin (Anti‐Mb), anti‐hemoglobin (Anti‐Hb) and anti‐gliadin respectively. The immobilizations of myoglobin, hemoglobin and gliadin onto Fullerene C60 were studied with a C60‐coated piezoelectric crystal detection system, respectively. The partially irreversible frequency responses for theses proteins were observed by a desorption study, implying that C60 can strongly adsorb these proteins. Thus, immobilized C60‐Mb, C60‐Hb and C60‐gliadin coating materials were successfully prepared and identified with FTIR spectrometry. The C60‐Mb, C60‐Hb and C60‐gliadin coated piezoelectric (PZ) quartz crystal immunosensors with homemade computer interfaces for signal acquisition and data processing were developed and applied for detection of Anti‐Mb, Anti‐Hb and anti‐gliadin respectively. The C60‐protein coated PZ immunosensors for Anti‐Mb, Anti‐Hb and antigliadin exhibited linear frequency responses to the concentrations of theses anti‐proteins with sensitivities of 1.43 × 10³, 2.59 × 10³ and 8.05 × 10³ Hz/(mg/mL) respectively. The detection limits of these PZ‐immunosensors were 4.36 × 10^?3, 3.23 × 10^?3 and 1.98 × 10^?3 mg/mL for Anti‐Mb, Anti‐Hb and anti‐gliadin respectively. Effects of pH and temperature on the frequency responses of the anti‐protein PZ‐immunosensors were also investigated. The optimum pH of these anti‐proteins and the optimum temperature for the PZ‐immunosensors were observed at pH = 7 and around 30 °C respectively. The interferences of various common species in human blood, e.g., cysteine, tyrosine, urea, glucose, ascorbic acid and metal ions, to these anti‐protein PZ‐immunosensors were also investigated respectively. These species showed nearly no interference or quite small interference with the anti‐protein PZ‐immunosensors. The reproducibility and lifetime of these immobilized C60‐protein coated PZ crystal immunosensors were also investigated and discussed.     

3β,7α,12α‐Tri­formyl­oxy‐24‐nor‐5β‐chol‐22‐ene

The title compound, alternatively called 24‐nor‐5β‐chol‐22‐ene‐3β,7α,12α‐triyl triformate, C₂₆H₃₈O₆, has a cis junction between two of the six‐membered rings. All three of the six‐membered rings have chair conformations that are slightly flattened and the five‐membered ring has a 13β,14α‐half‐chair conformation. The 3β, 7α and 12α ring substituents are axial and the 17β group is equatorial. The 3β‐formyl­oxy group is involved in one weak intermol­ecular C—H⋯O bond, which links the mol­ecules into dimers in a head‐to‐head fashion.     

Iron‐Catalyzed C−H Activation with Propargyl Acetates: Mechanistic Insights into Iron(II) by Experiment,Kinetics, Mössbauer Spectroscopy,and Computation

An iron‐catalyzed C?H/N?H alkyne annulation was realized by using a customizable clickable triazole amide under exceedingly mild reaction conditions. A unifying mechanistic approach combining experiment, spectroscopy, kinetics, and computation provided strong support for facile C?H activation by a ligand‐to‐ligand hydrogen transfer (LLHT) mechanism. Combined Mössbauer spectroscopic analysis and DFT calculations were indicative of high‐spin iron(II) species as the key intermediates in the C?H activation manifold.     

Nanopore label‐free detection of single‐nucleotide deletion in Baxα/BaxΔ2

Baxα, a key tumor suppressor gene, will not be expressed correctly as a result of single nucleotide mutation in its microsatellite region; Instead, BaxΔ2, an isoform of Baxα, is often produced. In addition, lack of the exon 2 due to an alternative splicing, BaxΔ2 has the same sequence as Baxα except single base deletion from eight continuous guanines (G8) to G7. Most of the currently available methods for Bax∆2 detection are inefficient and time‐consuming, and/or require the use of labels or dyes. In this work, we reported a label‐free nanopore sensing strategy to differentiate between Baxα and BaxΔ2 with a DNA polymer as a molecular probe based on alternative spliced sequences. Two DNA molecules were designed to selectively detect Baxα and BaxΔ2, respectively. The method was rapid, accurate, and highly sensitive: picomolar concentrations of target nucleic acids could be detected in minutes. Our developed simple and fast nanopore‐based detection strategy is not only useful for distinguishing between Baxα and Bax∆2, but also provides a useful tool for detection of other single‐base mutations in genetic diagnosis.     

Intra‐ and Intermolecular H‐Bonds of Alcohols in DMSO, 1H‐NMR Analysis of Inter‐Residue H‐Bonds in Selected Oligosaccharides: Cellobiose,Lactose, N,N′‐Diacetylchitobiose,Maltose, Sucrose,Agarose, and Hyaluronates

Inter‐residue H‐bonds of oligosaccharides in (D₆)DMSO have been assigned on the basis of a combined interpretation of the chemical shift (δ(OH)), coupling constant (J(H,OH)), and temperature dependence (Δδ(OH)/ΔT) of OH signals. Cellobiose, lactose, and N,N′‐diacetylchitobiose possess a completely persistent C(3)OH⋅⋅⋅OC(5′) H‐bond. Maltose is characterised by flip‐flop H‐bonds between HO−C(3) and HO−C(2′), and agarose by two weakly persistent inter‐residue H‐bonds. Sucrose forms an equilibrium of differently H‐bonded species, and hyaluronates possess four strong inter‐residue H‐bonds.     

Quantification, 2DE analysis and identification of enriched glycosylated proteins from mouse muscles: Difficulties and alternatives

One of the problems with 2DE is that proteins present in low amounts in a sample are usually not detected, since their signals are masked by the predominant proteins. The elimination of these abundant proteins is not a guaranteed solution to achieve the desired results. The main objective of this study was the comparison of common and simple methodologies employed for 2DE analysis followed by MS identification, focusing on a pre‐purified sample using a wheat germ agglutinin (WGA) column. Adult male C57Black/Crj6 (C57BL/6) mice were chosen as the model animal in this study; the gastrocnemius muscles were collected and processed for the experiments. The initial fractionation with succinylated WGA was successful for the elimination of the most abundant proteins. Two quantification methods were employed for the purified samples, and bicinchoninic acid (BCA) was proven to be most reliable for the quantification of glycoproteins. The gel staining method, however, was found to be decisive for the detection of specific proteins, since their structures affect the interaction of the dye with the peptide backbone. The Coomassie Blue R‐250 dye very weakly stained the gel with the WGA purified sample. When the same gel was stained with silver nitrate, however, MS could positively assign 12 new spots. The structure of the referred proteins was not found to be prone to interaction with Coomassie blue.     

The effect of antagonists on the conformational exchange of the retinoid X receptor alpha ligand‐binding domain

The effect of retinoid X receptor (RXR) antagonists on the conformational exchange of the RXR ligand‐binding domain (LBD) remains poorly characterized. To address this question, we used nuclear magnetic resonance spectroscopy to compare the chemical shift perturbations induced by RXR antagonists and agonists on the RXRα LBD when partnered with itself as a homodimer and as the heterodimeric partner with the peroxisome proliferator‐activated receptor γ (PPARγ) LBD. Chemical shift mapping on the crystal structure showed that agonist binding abolished a line‐broadening effect caused by a conformational exchange on backbone amide signals for residues in helix H3 and other regions of either the homo‐ or hetero‐dimer, whereas binding of antagonists with similar binding affinities failed to do so. A lineshape analysis of a glucocorticoid receptor‐interacting protein 1 NR box 2 coactivator peptide showed that the antagonists enhanced peptide binding to the RXRα LBD homodimer, but to a lesser extent than that enhanced by the agonists. This was further supported by a lineshape analysis of the RXR C‐terminal residue, threonine 462 (T462) in the homodimer but not in the heterodimer. Contrary to the agonists, the antagonists failed to abolish a line‐broadening effect caused by a conformational exchange on the T462 signal corresponding to the RXRα LBD–antagonist–peptide ternary complex. These results suggest that the antagonists lack the ability of the agonists to shift the equilibrium of multiple RXRα LBD conformations in favor of a compact state, and that a PPARγ LBD‐agonist complex can prevent the antagonist from enhancing the RXRα LBD‐coactivator binding interaction. Copyright © 2009 John Wiley & Sons, Ltd.     

Studying the effect of microwave heating on the digestion process and identification of proteins

The impact of microwave irradiation on the in‐solution digestion processes and the detection limit of proteins are systematically studied. Kinetic processes of many peptides produced through the trypsin digestion of various proteins under microwave heating at 50°C were investigated with MALDI‐MS. This study also examines the detection limits and digestion completeness of individual proteins under microwave heating at 50°C and at different time intervals (1, 5 and 30 min) using LC‐MS. We conclude that if the peptides without missed cleavage dictate the detection limit, conventional digestion will lead to a better detection limit. The detection limit may not differ between the microwave and conventional heating if the peptides with missed cleavage sites and strong intensity are formed at the very early stage (i.e., less than 1 min) and are not further digested throughout the entire digestion process. The digestion of Escherichia coli lysate was compared under conventional and short time (microwave) conditions. The number of proteins identified under conventional heating exceeded that obtained from microwave heating over heating periods less than 5 min. The overall results show that the microwave‐assisted digestion is not complete. Although the sequence coverage might be better, the detection limit might be worse than that under conventional heating.