title: Hydrogen-bond detection, configuration assignment and rotamer correction of side-chain amides in large proteins by NMR spectroscopy through protium/deuterium isotope effects.
abstract: The configuration and hydrogen-bonding network of side-chain amides in a 35 kDa protein were determined by measuring differential and trans-hydrogen-bond H/D isotope effects by using the isotopomer-selective (IS)-TROSY technique, which leads to a reliable recognition and correction of erroneous rotamers that are frequently found in protein structures. First, the differential two-bond isotope effects on carbonyl (13)C' shifts, which are defined as Delta(2)Delta(13)C'(ND) = (2)Delta(13)C'(ND(E))-(2)Delta(13)C'(ND(Z)), provide a reliable means for the configuration assignment for side-chain amides, because environmental effects (hydrogen bonds and charges, etc.) are greatly attenuated over the two bonds that separate the carbon and hydrogen atoms, and the isotope effects fall into a narrow range of positive values. Second and more importantly, the significant variations in the differential one-bond isotope effects on (15)N chemical shifts, which are defined as Delta(1)Delta(15)N(D) = (1)Delta(15)N(D(E))-(1)Delta(15)N(D(Z)) can be correlated with hydrogen-bonding interactions, particularly those involving charged acceptors. The differential one-bond isotope effects are additive, with major contributions from intrinsic differential conjugative interactions between the E and Z configurations, H-bonding interactions, and charge effects. Furthermore, the pattern of trans-H-bond H/D isotope effects can be mapped onto more complicated hydrogen-bonding networks that involve bifurcated hydrogen-bonds. Third, the correlations between Delta(1)Delta(15)N(D) and hydrogen-bonding interactions afford an effective means for the correction of erroneous rotamer assignments of side-chain amides. Rotamer correction by differential isotope effects is not only robust, but also simple and can be applied to large proteins.
authors.affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA. liua@msu.edu
authors.forename: Aizhuo
authors.initials: A
authors.lastname: Liu
authors.forename: Jifeng
authors.initials: J
authors.lastname: Wang
authors.forename: Zhenwei
authors.initials: Z
authors.lastname: Lu
authors.forename: Lishan
authors.initials: L
authors.lastname: Yao
authors.forename: Yue
authors.initials: Y
authors.lastname: Li
authors.forename: Honggao
authors.initials: H
authors.lastname: Yan
mesh_terms: D000577:Amides / Q000737:chemistry*; D043525:Cytosine Deaminase / Q000737:chemistry*; D003903:Deuterium; D006860:Hydrogen Bonding; D007553:Isotope Labeling; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D011522:Protons; D015003:Yeasts / Q000201:enzymology
chemical_list: D000577:Amides; D009587:Nitrogen Isotopes; D011522:Protons; D003903:Deuterium; D043525:Cytosine Deaminase