13 C-NMR studies on disulfide bond isomerization in bovine pancreatic trypsin inhibitor (BPTI)

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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 Cys14â??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 13C spectra were recorded at small temperature intervals for BPTI samples selectively labeled with site-specifically 13C-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 Cys14â??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 13C relaxation dispersion experiments was obtained through the carbon line-shape profile. With respect to the m c38 isomerization, the 1H-13C signals at the β-position of the minor state were resolved from the major peaks and detected by exchange experiments at a low temperature.



Source: Journal of Biomolecular NMR