39 research outputs found
Ile, Leu, and Val Methyl Assignments of the 723-Residue Malate Synthase G Using a New Labeling Strategy and Novel NMR Methods
Side Chain Assignments of Ile δ1 Methyl Groups in High Molecular Weight Proteins: an Application to a 46 ns Tumbling Molecule
Precision Measurements of Deuterium Isotope Effects on the Chemical Shifts of Backbone Nuclei in Proteins: Correlations with Secondary Structure
Precision NMR measurements of deuterium isotope effects
on the
chemical shifts of backbone nuclei in proteins (<sup>15</sup>N, <sup>13</sup>CO, <sup>13</sup>C<sub>α</sub>, and <sup>1</sup>HN)
arising from <sup>1</sup>H-to-<sup>2</sup>H substitutions at aliphatic
carbon sites. Isolation of molecular species with a defined protonation/deuteration
pattern at carbon-α/β positions allows distinguishing
and accurately quantifying different isotope effects within the protein
backbone. The isotope shifts measured in the partially deuterated
protein ubiquitin are interpreted in terms of backbone geometry via
empirical relationships describing the dependence of isotope shifts
on (φ; ψ) backbone dihedral angles. Because of their relatively
large magnitude and clear dependence on the protein secondary structure,
the two- and three-bond backbone amide <sup>15</sup>N isotope shifts, <sup>2</sup>ΔNÂ(C<sub>α,i</sub>D) and <sup>3</sup>ΔNÂ(C<sub>α,i‑1</sub>D), can find utility for NMR structural refinement
of small-to-medium size proteins
Estimating Side-Chain Order in [U‑<sup>2</sup>H;<sup>13</sup>CH<sub>3</sub>]‑Labeled High Molecular Weight Proteins from Analysis of HMQC/HSQC Spectra
A simple
approach for quantification of methyl-containing side-chain
mobility in high molecular weight methyl-protonated, uniformly deuterated
proteins is described, based on the measurement of peak intensities
in methyl <sup>1</sup>H–<sup>13</sup>C HMQC and HSQC correlation
maps and relaxation rates of slowly decaying components of methyl <sup>1</sup>H–<sup>13</sup>C multiple-quantum coherences. A strength
of the method is that [U-<sup>2</sup>H;<sup>13</sup>CH<sub>3</sub>]-labeled protein samples are required that are typically available
at an early stage of any analysis. The utility of the methodology
is demonstrated with applications to three protein systems ranging
in molecular weight from 82 to 670 kDa. Although the approach is only
semiquantitative, a high correlation between order parameters extracted
via this scheme and other more established methods is nevertheless
demonstrated
Selective detection of 13CHD2 signals from a mixture of 13CH3/13CH2D/ 13CHD2 methyl isotopomers in proteins
In NMR spectra of partially deuterated proteins methyl correlations are commonly observed as a combination of signals from 13CH3, 13CH2D and 13CHD2 isotopomers. In a number of NMR applications, methyl groups of the 13CHD2 variety are targeted because of their AX-like character and concomitant simplification of the involved relaxation mechanisms. Although complete elimination of signals from 13CH2D methyl groups can be easily achieved in such applications, if the magnetization is not transferred through deuterium nuclei, efficient suppression of usually stronger 13CH3 peaks is more problematic. A pair of simple pulse-scheme elements are presented that achieve almost complete suppression of 13CH3 signals in the mixtures of 13CH 3/13CH2D/13CHD2 methyl isotopomers of small proteins at the expense of a moderate (~20-to-40%) reduction in intensities of the targeted 13CHD2 groups. The approaches described are based purely on scalar coupling (1J CH) evolution properties of different 13C and 1H transitions within 13CH3 spin-systems and are superior to magnetization transfer through deuterons with respect to sensitivity of the detected 13CHD2 methyl signals. ? 2010 Elsevier Inc. All rights reserved