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HSQC Experiments (hsqc + experiment)
Selected AbstractsCell-free expression and selective isotope labelling in protein NMRMAGNETIC RESONANCE IN CHEMISTRY, Issue S1 2006David Staunton Abstract Isotope labelling is a very powerful tool in NMR studies of proteins and has been employed in various ways for over 40 years. 15N and 13C incorporation, using recombinant expression systems, is now commonplace because heteronuclear experiments assist with the fundamental problems of peak resolution and assignment. The use of selective labelling for peak assignment has been restricted by the scrambling of isotope label through metabolic pathways within the expression host organism. The availability of efficient cell-free expression systems with low levels of metabolic conversion allow the increasing use of selective isotope labelling as a tool in protein NMR. We describe two examples, one where a selective labelling scheme can identify backbone amide peaks from unassigned 1H15N HSQC and HNCO spectra of a 84 residue protein, and another where a specific backbone amide in a 198 residue construct of the ninth and tenth Type III repeats from human fibronectin can be labelled and rapidly identified using a simple HSQC experiment. Copyright © 2006 John Wiley & Sons, Ltd. [source] Simultaneous determination of 1H,1H and 1H,13C residual dipolar couplings in a chiral liquid crystal solvent using a natural abundance HSQC experimentMAGNETIC RESONANCE IN CHEMISTRY, Issue 7 2005Vasilios M. Marathias Abstract A high-resolution, phase-sensitive, natural abundance F2 -coupled 1H,13C HSQC (F2HSQC) NMR experiment was developed to measure simultaneously both nDHH and 1DCH residual dipolar couplings (RDCs) of small molecules present in a chiral polypeptide liquid crystal solvent system composed of poly-,-benzyl- L -glutamate (PBLG) in CDCl3. Because this is an indirect-detection NMR experiment, the relatively small amount of sample (7.5 mg in this study) and short acquisition times (5 h) that are required make this HSQC experiment well suited for samples that are either limited in solubility or in quantity or require short analysis times. The F2HSQC experiment can be performed without any specialized equipment or sample modification and can enhance our ability to measure RDCs accurately and rapidly in polypeptide liquid crystal solvents. Copyright © 2005 John Wiley & Sons, Ltd. [source] The identity of the O-specific polysaccharide structure of Citrobacter strains from serogroups O2, O20 and O25 and immunochemical characterisation of C. youngae PCM 1507 (O2a,1b) and related strainsFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 1-2 2003gorzata Miesza Abstract Serological studies using SDS,PAGE and immunoblotting revealed that from five strains that are ascribed to Citrobacter serogroup O2, four strains, PCM 1494, PCM 1495, PCM 1496 and PCM 1507, are reactive with specific anti- Citrobacter O2 serum. In contrast, strain PCM 1573 did not react with anti- Citrobacter O2 serum and, hence, does not belong to serogroup O2. The LPS of Citrobacter youngae O2a,1b (strain PCM 1507) was degraded under mild acidic conditions and the O-specific polysaccharide (OPS) released was isolated by gel chromatography. Sugar and methylation analyses along with 1H- and 13C-NMR spectroscopy, including two-dimensional 1H,1H COSY, TOCSY, NOESY and 1H,13C HSQC experiments, showed that the repeating unit of the OPS has the following structure: NMR spectroscopic studies demonstrated that Citrobacter werkmanii O20 and C. youngae O25 have the same OPS structure as C. youngae O2. Sugar and methylation analyses of the core oligosaccharide fractions demonstrated structural differences in the lipopolysaccharide core regions of these strains, which may substantiate their classification in different serogroups. [source] 1H,13C and 19F NMR data of N -substituted 6-(4-methoxyphenyl)-7H -pyrrolo[2,3- d]pyrimidin-4-amines in DMSO- d6MAGNETIC RESONANCE IN CHEMISTRY, Issue 3 2010Christopher Sørum Abstract Chemical shift assignment of seven N -substituted 6-(4-methoxyphenyl)-7H -pyrrolo[2, 3- d]pyrimidin-4-amines, six of which are fluorinated, have been performed based on 1H, 13C, 19F, and 2D COSY, HMBC and HSQC experiments. Copyright © 2009 John Wiley & Sons, Ltd. [source] High resolution in heteronuclear 1H,13C NMR experiments by optimizing spectral aliasing with one-dimensional carbon dataMAGNETIC RESONANCE IN CHEMISTRY, Issue 1 2003Damien Jeannerat Abstract In the chemistry literature it is common to provide NMR data on both proton and carbon spectra based on one-dimensional experiments, but often only proton spectra are assigned. The absence of a complete attribution of the carbons is in good part due to the difficulty in reaching the necessary resolution in the carbon dimension of two-dimensional experiments. It has already been shown that high-resolution heteronuclear spectra can be acquired within nearly the same acquisition time using a violation of the Nyquist condition. For a spectral width reduction by a given factor k, the resolution increases by the same factor as long as it is not limited by relaxation. The price to pay for such an improvement is a k -fold ambiguity in the chemical shift of the signal along the folded or aliased dimension. The computer algorithm presented in this paper takes advantage of the peak list stemming from one-dimensional spectra in order to calculate spectral widths for which the ambiguities in the aliased dimension of heteronuclear experiments are eliminated or at least minimized. The resolution improvement factor is only limited by the natural lineshape and reaches a typical value higher than 100. The program may be set to run automatically on spectrometers equipped with automatic sample changers. Applications to short-range HSQC experiments and long-range HMBC spectra of steroids, carbohydrates, a peptide and a mixture of isomers are shown as examples. Copyright © 2002 John Wiley & Sons, Ltd. [source] Polymerization of the SAM domain of MAPKKK Ste11 from the budding yeast: Implications for efficient signaling through the MAPK cascadesPROTEIN SCIENCE, Issue 3 2005Surajit Bhattacharjya Abstract The sterile ,-motif (SAM) is a protein module ,70 residues long and mainly involved in the protein,protein interactions of cell signaling and transcriptional repression. The SAM domain of the yeast MAPKKK Ste11 has a well-folded dimeric structure in solution. Interestingly, the well-folded dimer of the Ste11 SAM undergoes a time-dependent self-assembly upon lowering of the pH, leading to the formation of high molecular weight oligomers. The oligomeric structures rapidly disassemble to the well-folded dimer upon reversal of the pH to close to neutral conditions. Circular dichroism (CD) and atomic force microscopy (AFM) experiments demonstrate that the oligomeric structure formed at pH 5.0 appears to be highly helical and has architecture akin to proto-fibrils. Residue-specific kinetics of pH-triggered oligomerization obtained from real-time 15N- 1H HSQC experiments indicate that the dimer-oligomer transition appears to involve all residues of the well-folded dimeric structure of the Ste11 SAM. Very interestingly, the interactions of the Ste11 and Ste50 SAM domains also lead to the formation of non-homogeneous hetero-complexes with significant populations of high molecular weight aggregates. AFM imaging shows that the Ste11-Ste50 hetero-polymeric aggregates assume the shapes of circular nano-particles with dimensions of 50,60 nano-meters (nm), in contrast to the proto-fibrils formed by the Ste11 SAM domain alone. Such intrinsic propensity for dimer to oligomer transition of the Ste50-binding SAM domain of Ste11 may endow the MAPKKK Ste11 with unique functional properties required for efficient and high fidelity signal transduction in the budding yeast. [source] Study of Structural Stability of Cyclophilin A by NMR and Circular Dichroism SpectraCHINESE JOURNAL OF CHEMISTRY, Issue 7 2006Yan-Hong Shi Abstract The structural stability of cyclophilin A (CypA) was investigated using H/D exchange and temperature coefficients of chemical shifts of amide protons, monitored by 2D heteronuclear NMR spectroscopy. Amide proton exchange rates were measured by H/D exchange experiments for slow-exchange protons and measured by SEA (Solvent Exposed Amides)-HSQC experiments for fast-exchange protons. Temperature coefficients of chemical shifts and hydrogen exchange rates of amide protons show reasonably good correlation with the protein structure. Totally, 44 out of 153 non-proline assigned residues still exist in 86 d of hydrogen-deuterium exchange at 4 °C, suggesting that CypA structure should be highly stable. Residues in secondary structures of ,2, ,1, ,2, ,5, ,6 and ,7 might constitute the hydrophobic core of the protein. The change in free energy of unfolding (,Gu) of CypA was estimated to be (21.99±1.53) kJ·mol,1 by circular dichroism (CD). The large free energy change is also an indicator of the high structural stability. [source] | ||||||||||