Refolding of β‐lactoglobulin studied by stopped‐flow circular dichroism at subzero temperatures

FEBS Letters

Shimada, Lui²  Arai, Munehito⁴  Nakagawa, Tatsuo³  Zhou, Jun-Mei¹  Hu, Dong-mei²  Kihara, Hiroshi²  Qin, Zhi-jie² 

[1] National Laboratory of Macromolecules, Institute of Biophysics, Academia Sinica, 15 Datun Road, Beijing 100101, PR China;Department of Physics, Kansai Medical University, 18-89 Uyama-Higashi, Hirakata 573-1136, Japan;Unisoku Inc., 2-4-3 Kasugano, Hirakata 573-0131, Japan;Department of Physics, School of Science, University of Tokyo, 7-3-1 Hongo, Tokyo 113-0033, Japan

关键词: GuHCl;    guanidine hydrochloride;    PBS;    phosphate-buffered saline;    T-jump;    temperature jump;    CD;    circular dichroism;   

DOI  :  10.1016/S0014-5793(01)02886-1

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Refolding of bovine β-lactoglobulin was studied by stopped-flow circular dichroism at subzero temperatures. In ethylene glycol 45%–buffer 55% at −15°C, the isomerization rate from the kinetic intermediate rich in α-helix to the native state is approximately 300-fold slower than that at 4°C in the absence of ethylene glycol, whereas the initial folding is completed within the dead time of the stopped-flow apparatus (10 ms). At −28°C, we observed at least three phases; the fastest process, accompanied by an increase of α-helix content, is completed within the dead time of the stopped-flow apparatus (10 ms), the second phase, accompanied by an increase of α-helix content with the rate of 2 s⁻¹, and the third phase, accompanied by a decrease of α-helix content. This last phase, corresponding to the isomerization process at −15°C described above, was so slow that we could not monitor any changes within 4 h. Based on the findings above, we propose that rapid α-helix formation and their concurrent collapse are common even in proteins rich in β-structure in their native forms.

【 授权许可】

Unknown