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Graesser DT Wylie BJ Nieuwkoop AJ Franks WT Rienstra CM 《Magnetic resonance in chemistry : MRC》2007,45(Z1):S129-S134
We present a novel rotational-echo double resonance (REDOR) method for detection of multiple (19)F-(15)N distances in solid proteins. The method is applicable to protein samples containing a single (19)F label, in addition to high levels of (13)C and (15)N enrichment. REDOR dephasing pulses are applied on the (19)F channel during an indirect constant time chemical shift evolution period on (15)N, and polarization is then transferred to (13)C for detection, with high-power (1)H decoupling throughout the sequence. This four-channel experiment reports site-specifically on (19)F-(15)N distances, with highly accurate determinations of approximately 5 A distances and detection of correlations arising from internuclear distances of at least 8 A. We demonstrate the method on the well-characterized 56-residue model protein GB1, where the sole tryptophan residue (Trp-43) has been labeled with 5-(19)F-Trp, in a bacterial growth medium also including (13)C-glucose and (15)N ammonium chloride. In GB1, 11 distances are determined, all agreeing within 20% of the X-ray structure distances. We envision the experiment will be utilized to measure quantitative long-range distances for protein structure determination. 相似文献
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CM Silva MF Duarte ML Mira MH Florêncio K Versluis AJ Heck 《Rapid communications in mass spectrometry : RCM》1999,13(12):1098-1103
Fast atom bombardment, combined with high-energy collision-induced tandem mass spectrometry, has been used to investigate gas-phase metal-ion interactions with captopril, enalaprilat and lisinopril, all angiotensin-converting enzyme inhibitors.Suggestions for the location of metal-binding sites are presented. For captopril, metal binding occurs most likely at both the sulphur and the nitrogen atom. For enalaprilat and lisinopril, binding preferably occurs at the amine nitrogen. Copyright 1999 John Wiley & Sons, Ltd. 相似文献
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Ramautar R Mayboroda OA Derks RJ van Nieuwkoop C van Dissel JT Somsen GW Deelder AM de Jong GJ 《Electrophoresis》2008,29(12):2714-2722
A capillary electrophoresis-time of flight-mass spectrometry (CE-TOF-MS) method for the analysis of amino acids in human urine was developed. Capillaries noncovalently coated with a bilayer of Polybrene (PB) and poly(vinyl sulfonate) (PVS) provided a considerable EOF at low pH, thus facilitating the fast separation of amino acids using a BGE of 1 M formic acid (pH 1.8). The PB-PVS coating proved to be very consistent yielding stable CE-MS patterns of amino acids in urine with favorable migration time repeatability (RSDs <2%). The relatively low sample loading capacity of CE was circumvented by an in-capillary preconcentration step based on pH-mediated stacking allowing 100-nL sample injection (i.e. ca. 4% of capillary volume). As a result, LODs for amino acids were down to 20 nM while achieving satisfactory separation efficiencies. Preliminary validation of the method with urine samples showed good linear responses for the amino acids (R(2) >0.99), and RSDs for peak areas were <10%. Special attention was paid to the influence of matrix effects on the quantification of amino acids. The magnitude of ion suppression by the matrix was similar for different urine samples. The CE-TOF-MS method was used for the analysis of urine samples of patients with urinary tract infection (UTI). Concentrations of a subset of amino acids were determined and compared with concentrations in urine of healthy controls. Furthermore, partial least squares-discriminant analysis (PLS-DA) of the CE-TOF-MS dataset in the 50-450 m/z region showed a distinctive grouping of the UTI samples and the control samples. Examination of score and loadings plot revealed a number of compounds, including phenylalanine, to be responsible for grouping of the samples. Thus, the CE-TOF-MS method shows good potential for the screening of body fluids based on the analysis of endogenous low-molecular weight metabolites such as amino acids and related compounds. 相似文献
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Dr. Ümit Akbey Dr. Andrew J. Nieuwkoop Dr. Sebastian Wegner Anja Voreck Dr. Britta Kunert Dr. Priyanga Bandara Dr. Frank Engelke Prof. Dr. Niels Chr. Nielsen Prof. Dr. Hartmut Oschkinat 《Angewandte Chemie (International ed. in English)》2014,53(9):2438-2442
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. 相似文献
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