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Backbone Amide (backbone + amide)

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Chemistry100%

Selected Abstracts

Cell-free expression and selective isotope labelling in protein NMR

MAGNETIC RESONANCE IN CHEMISTRY, Issue S1 2006
David 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]

Fourier transform vibrational circular dichroism as a decisive tool for conformational studies of peptides containing tyrosyl residues

BIOPOLYMERS, Issue 1 2003
Attila Borics
Abstract Previous UV,circular dichroism (UV,CD) and NMR studies showed that Ac-AAAAAAAEAAKA-NH2 has an ,-helical structure in 50% (v/v) aqueous trifluoroethanol. Replacement of Ala1 to Ala6 with Tyr results in spectra that show an apparent loss of helicity in the same solvent. This apparent loss of helicity could be attributed to the coupling of the tyrosyl side chain chromophore with the backbone amide. However, such electronic coupling does not affect the vibrational CD (VCD) spectra. The VCD spectra of the peptides with tyrosyl residues were identical to that of the peptide containing no Tyr, which shows the same ,-helical structure. Because it is now clear that Tyr replacement does not change the backbone conformation of peptides, UV,CD measurements should be complemented by VCD to determine the secondary structure when electronic effects can disturb the UV,CD spectrum of the inherent structure. © 2002 Wiley Periodicals, Inc. Biopolymers (Biospectroscopy) 72: 21,24, 2003 [source]

Response of native and denatured hen lysozyme to high pressure studied by 15N/1H NMR spectroscopy

FEBS JOURNAL, Issue 6 2001
Yuji O. Kamatari
High-pressure 15N/1H NMR techniques were used to characterize the conformational fluctuations of hen lysozyme, in its native state and when denatured in 8 m urea, over the pressure range 30,2000 bar. Most 1H and 15N signals of native lysozyme show reversible shifts to low field with increasing pressure, the average pressure shifts being 0.069 ± 0.101 p.p.m. (1H) and 0.51 ± 0.36 p.p.m. (15N). The shifts indicate that the hydrogen bonds formed to carbonyl groups or water molecules by the backbone amides are, on average, shortened by ,,0.02 Å as a result of pressure. In native lysozyme, six residues in the , domain or at the ,/, domain interface have anomalously large nonlinear 15N and 1H chemical-shift changes. All these residues lie close to water-containing cavities, suggesting that there are conformational changes involving these cavities, or the water molecules within them, at high pressure. The pressure-induced 1H and 15N shifts for lysozyme denatured in 8 m urea are much more uniform than those for native lysozyme, with average backbone amide shifts of 0.081 ± 0.029 p.p.m. (1H) and 0.57 ± 0.14 p.p.m. (15N). The results show that overall there are no significant variations in the local conformational properties of denatured lysozyme with pressure, although larger shifts in the vicinity of a persistent hydrophobic cluster indicate that interactions in this part of the sequence may rearrange. NMR diffusion measurements demonstrate that the effective hydrodynamic radius of denatured lysozyme, and hence the global properties of the denatured ensemble, do not change detectably at high pressure. [source]

Effects of electrospray capillary temperature on amide hydrogen exchange

RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 9 2008
Stephen J. Coales
Amide hydrogen/deuterium (H/D) exchange coupled with proteolysis, high-perfeomance liquid chromatographic (HPLC) separation and mass spectrometry (MS) has become a powerful tool to study protein dynamics in solution. Prior to the execution of H/D exchange experiments, various experimental parameters have to be set, including proteolysis, HPLC, and MS conditions. Here we investigate the effects of electrospray capillary temperature on deuterium retention in backbone amides of various pepsin-generated cytochrome c peptides. Lower capillary temperature generally helps retain more deuterium than higher capillary temperature. When the capillary temperature was 150°C, on average 26% more deuterium was retained than when the capillary temperature was set at 250°C. The effects of capillary temperature varied depending on the ions monitored. There was little difference in deuterium retention among different charge state species of the same peptide at 150°C. However, a lower charge state ion loses more deuterium atoms going from 150°C to 250°C than the corresponding higher charge state species. These results indicate that the capillary temperature should be optimized not only to maximize the signal-to-noise of each ion followed in H/D exchange experiments, but also to minimize the deuterium loss of the ions. Also the loss of deuterium in several ions, especially lower charge state ones, should be monitored in the optimization, as the temperature effects vary among ions and are more significant for lower charge state ions. Copyright © 2008 John Wiley & Sons, Ltd. [source]