title: (13)C-NMR studies on disulfide bond isomerization in bovine pancreatic trypsin inhibitor (BPTI).
abstract: Conformational isomerization of disulfide bonds is associated with the dynamics and thus the functional aspects of proteins. However, our understanding of the isomerization is limited by experimental difficulties in probing it. We explored the disulfide conformational isomerization of the Cys14-Cys38 disulfide bond in bovine pancreatic trypsin inhibitor (BPTI), by performing an NMR line-shape analysis of its Cys carbon peaks. In this approach, 1D (13)C spectra were recorded at small temperature intervals for BPTI samples selectively labeled with site-specifically (13)C-enriched Cys, and the recorded peaks were displayed in the order of the temperature after the spectral scales were normalized to a carbon peak. Over the profile of the line-shape, exchange broadening that altered with temperature was manifested for the carbon peaks of Cys14 and Cys38. The Cys14-Cys38 disulfide bond reportedly exists in equilibrium between a high-populated (M) and two low-populated states (m c14 and m c38). Consistent with the three-site exchange model, biphasic exchange broadening arising from the two processes was observed for the peak of the Cys14 α-carbon. As the exchange broadening is maximized when the exchange rate equals the chemical shift difference in Hz between equilibrating sites, semi-quantitative information that was useful for establishing conditions for (13)C relaxation dispersion experiments was obtained through the carbon line-shape profile. With respect to the m c38 isomerization, the (1)H-(13)C signals at the β-position of the minor state were resolved from the major peaks and detected by exchange experiments at a low temperature.
authors: Structural Biology Research Center, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8602, Japan.|Department of Structural BioImaging, Faculty of Life Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto, 862-0973, Japan.
authors: Mitsuhiro
authors: M
authors: Takeda
authors: Structural Biology Research Center, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8602, Japan.
authors: Yohei
authors: Y
authors: Miyanoiri
authors: Graduate School of Science and Engineering, Tokyo Metropolitan University, 1-1 Minami-ohsawa, Hachioji, Tokyo, 192-0397, Japan.|SAIL Technologies Inc., 2008-2 Wada, Tama, Tokyo, 206-0001, Japan.
authors: Tsutomu
authors: T
authors: Terauchi
authors: Structural Biology Research Center, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8602, Japan. kainosho@tmu.ac.jp.|Graduate School of Science and Engineering, Tokyo Metropolitan University, 1-1 Minami-ohsawa, Hachioji, Tokyo, 192-0397, Japan. kainosho@tmu.ac.jp.
authors: Masatsune
authors: M
authors: Kainosho
mesh_terms: D000465:Algorithms; D000818:Animals; D007611:Aprotinin / Q000737:chemistry*; D066241:Carbon-13 Magnetic Resonance Spectroscopy / Q000379:methods; D002417:Cattle; D004220:Disulfides / Q000737:chemistry*; D007527:Isoenzymes; D008956:Models, Chemical; D015394:Molecular Structure; D050505:Mutant Proteins; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D013696:Temperature; D013816:Thermodynamics
chemical_list: D004220:Disulfides; D007527:Isoenzymes; D050505:Mutant Proteins; D007611:Aprotinin
keywords: 13C NMR; Disulfide bond isomerization; Exchange broadening; Site-specific 13C enrichment