An Empirical Correlation between Amide Deuterium Isotope Effects on 13CR Chemical Shifts and Protein Backbone Conformation

Three-dimensional, triple resonance NMR techniques are described for measurement of two-bond (intraresidual) and three-bond (sequential) amide deuterium isotope effects on 13CR chemical shifts. Measurements were carried out for uniformly 15N and 13C labeled human ubiquitin equilibrated in a 50% H2O/50% D2O mixed solvent. The three-bond isotope shift, 3∆CR(ND), ranges from about 10-50 ppb, and, except for residues with positive φ angles and residues followed by Gly, its magnitude is described by 3∆CR(ND) ) 30.1 + 22.2 sin(ψ) ( 3.4 ppb. The two-bond isotope shift, 2∆CR(ND), ranges from 70 to 116 ppb and is also dominated by the local backbone geometry at the CR position: 2∆CR(ND) ) 93.1 + 10.1 sin(φ+62°) + 12.0 sin(ψ+42°) ( 4.1 ppb. Deuterium isotope effects on chemical shifts in NMR have been extensively studied over the past two decades1-13 and have proven to be useful for spectral assignment and structure determination of small organic molecules. It is now generally established that isotope shift effects are vibrational in origin: the lower zero-point vibrational energy of the heavier isotopomer and the anharmonicity of the bond stretching potential curve results in a shorter average internuclear separation of the heavier isotopomer.7,10 The magnitude of the isotope effect on nuclear magnetic shielding is governed by a variety of factors, including the number of bonds between the observed nucleus and the position of the isotopic substitution, conformation, hybridization, substituents, and hydrogen bonding. However, understanding of these factors remains incomplete, especially concerning effects over more than two bonds. Reasonable agreement between experimentally observed isotope effects and ab initio results so far has only been obtained for very simple model compounds.7 An empirical treatment is therefore inevitable and may contribute to a better understanding of the physical origin of the effect. In previous empirical studies it was reported that the magnitude of the deuterium isotope effects over two bonds is related to the strength of the hydrogen bond donated by NH/D.11,12 It was also found that the three-bond isotope effects depend on the dihedral angle formed by the corresponding bonds, similar to a Karplus relationship.5,9,11 Taking advantage of the fact that NH protons in peptide bonds exchange, but relatively slowly, a deuterium isotope effect on the 13C chemical shifts of carbons in or near peptide bonds can readily be observed in molecules examined in a H2O/D2O solvent mixture. Applications have been reported for the carbonyl carbons in peptides1,14 and proteins.11,15,16 Recent renewed interest in deuteration of proteins has been fueled by its potential to yield longer 13C relaxation times, thereby extending the size limit of proteins that can be studied in detail by high resolution NMR.17-20 Although the intrinsic utility of one-bond 13CR-{1H/2H} deuterium isotope shifts, 1∆CR(D), for determining backbone geometry has been recognized,13 effects over two and three bonds generally are considered a nuisance as they broaden the width of 13C resonances in cases of incomplete deuteration. Variations observed in the magnitude of the isotope shifts also make it more difficult to correlate 13C chemical shifts in a protonated protein with those in the perdeuterated counterpart.21,22 However, with improved understanding of the origin of these isotope shifts, they potentially can become useful as reporters for side-