Journal
JOURNAL OF BIOMOLECULAR NMR
Volume 66, Issue 1, Pages 37-53Publisher
SPRINGER
DOI: 10.1007/s10858-016-0055-8
Keywords
Disulfide bond isomerization; C-13 NMR; Exchange broadening; Site-specific C-13 enrichment
Categories
Funding
- Ministry of Education, Culture, Sports, Science and Technology (MEXT)
- Japan Agency for Medical Research and development (AMED)
- Kurata Memorial Hitachi Science and Technology Foundation [21121002, 26119005, 25440018, 15K06966]
- Grants-in-Aid for Scientific Research [15K06966, 26119005] Funding Source: KAKEN
Ask authors/readers for more resources
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 C-13 spectra were recorded at small temperature intervals for BPTI samples selectively labeled with site-specifically C-13-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 alpha-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 C-13 relaxation dispersion experiments was obtained through the carbon line-shape profile. With respect to the m (c38) isomerization, the H-1-C-13 signals at the beta-position of the minor state were resolved from the major peaks and detected by exchange experiments at a low temperature.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available