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Conformational analysis by quantitative NOE measurements of the β-proton pairs across individual disulfide bonds in proteins

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Abstract

NOEs between the β-protons of cysteine residues across disulfide bonds in proteins provide direct information on the connectivities and conformations of these important cross-links, which are otherwise difficult to investigate. With conventional [U-13C, 15N]-proteins, however, fast spin diffusion processes mediated by strong dipolar interactions between geminal β-protons prohibit the quantitative measurements and thus the analyses of long-range NOEs across disulfide bonds. We describe a robust approach for alleviating such difficulties, by using proteins selectively labeled with an equimolar mixture of (2R, 3S)-[β-13C; α,β-2H2] Cys and (2R, 3R)-[β-13C; α,β-2H2] Cys, but otherwise fully deuterated. Since either one of the prochiral methylene protons, namely β2 (proS) or β3 (proR), is always replaced with a deuteron and no other protons remain in proteins prepared by this labeling scheme, all four of the expected NOEs for the β-protons across disulfide bonds could be measured without any spin diffusion interference, even with long mixing times. Therefore, the NOEs for the β2 and β3 pairs across each of the disulfide bonds could be observed at high sensitivity, even though they are 25% of the theoretical maximum for each pair. With the NOE information, the disulfide bond connectivities can be unambiguously established for proteins with multiple disulfide bonds. In addition, the conformations around disulfide bonds, namely χ2 and χ3, can be determined based on the precise proton distances of the four β-proton pairs, by quantitative measurements of the NOEs across the disulfide bonds. The feasibility of this method is demonstrated for bovine pancreatic trypsin inhibitor, which has three disulfide bonds.

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Acknowledgments

This work was supported by the Targeted Proteins Research Program (TPRP) to M. K. and partially by Grants-in-Aid for Young Scientists (B) (21770110, 23770109) to M. T. and a Grant-in-Aid in Innovative Areas (4104) to M. K.

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Authors and Affiliations

  1. Structural Biology Research Center, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8602, Japan

    Mitsuhiro Takeda & Masatsune Kainosho

  2. Center for Priority Areas, Graduate School of Science and Technology, Tokyo Metropolitan University, 1-1 Minami-ohsawa, Hachioji, 192-0397, Japan

    Tsutomu Terauchi & Masatsune Kainosho

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  1. Mitsuhiro Takeda
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  2. Tsutomu Terauchi
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Correspondence to Masatsune Kainosho.

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10858_2011_9587_MOESM1_ESM.doc

NOESY spectra (the region of the Cys5-Cys55 across-disulfide NOEs) of the UL-Cys-labeled BPTI, the l-[β-13C; α,β 2/β 3-2H2] Cys-labeled BPTI and the (l-[β-13C; α,β 2/β 3-2H2] Cys, [U-2H])-labeled BPTI at NOE mixing times of 50 and 400 ms and the comparison of their NOE build-up curves. The 1H T1 of β-protons of the Cys-labeled BPTI and the l-[β-13C; α,β 2/β 3-2H2] Cys-labeled protein in a deuterated background. The computation of inter-proton distances between Cys β-protons across a disulfide bond. The T1 and T2 values of Cys 13Cβ atoms in (l-[β-13C; α,β 2/β 3-2H2] Cys, [U-2H])-labeled BPTI. The cross-relaxation rates of the across-disulfide NOEs and the corresponding effective distances. These are provided in the supporting information. Supplementary material 1 (DOC 522 kb)

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Takeda, M., Terauchi, T. & Kainosho, M. Conformational analysis by quantitative NOE measurements of the β-proton pairs across individual disulfide bonds in proteins. J Biomol NMR 52, 127–139 (2012). https://doi.org/10.1007/s10858-011-9587-0

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