Abstract
An isotope labeling scheme is described in which specific protonation of methine and methyl protons of leucine and valine is obtained on a 15N/13C labeled background with uniform deuteration of all other non-exchangeable protons. The presence of a protonated methine group has little effect on the favorable relaxation properties of the methyl protons of Leu and Val. This labeling scheme permits the rotameric state of leucine side-chains to be readily determined by simple inspection of the pattern of Hγ(i)–HN(i) and Hγ(i)–HN(i+1) NOEs in a 3D 15N-separated NOE spectrum free of complications arising from spectral overlap and spin-diffusion. In addition, one-bond residual dipolar couplings for the methine 13C–1H bond vectors of Leu and Val can be accurately determined from an intensity J-modulated constant-time HCCH-COSY experiment and used to accurately orient the side-chains of Leu and Val. Incorporation of these data into structure refinement improves the accuracy with which the conformations of Leu and Val side-chains can be established. This is important to ensure optimal packing both within the protein core and at intermolecular interfaces. The impact of the method on protein structure determination is illustrated by application to enzyme IIAChitobiose, a 34 kDa homotrimeric phosphotransferase protein.
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Tang, C., Iwahara, J. & Clore, G.M. Accurate Determination of Leucine and Valine Side-chain Conformations using U-[15N/13C/2H]/[1H-(methine/methyl)-Leu/Val] Isotope Labeling, NOE Pattern Recognition, and Methine Cγ–Hγ/Cβ–Hβ Residual Dipolar Couplings: Application to the 34-kDa Enzyme IIAChitobiose. J Biomol NMR 33, 105–121 (2005). https://doi.org/10.1007/s10858-005-1206-5
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DOI: https://doi.org/10.1007/s10858-005-1206-5