A metal binding in the polypeptide chain improves the folding efficiency of a denatured and reduced protein

Takatoshi Ohkuri, Tadashi Ueda, Yuichiro Yoshida, Yoshito Abe, Naotaka Hamasaki, Taiji Imoto

研究成果: Contribution to journalArticle査読

抄録

In order to examine the effect of a metal binding to the polypeptide chain on the aggregation of a protein in the refolding process, we prepared a mutant hen lysozyme possessing the same Ca2+ binding site as in human a-lactalbumin by Escherichia coli expression system (Ser-1 CaB lysozyme). In the presence of 2 mM CaCl2, the refolding yield of Ser-1 CaB lysozyme at a low protein concentration (25 μg/mL) was similar to that of the wild-type lysozyme (80%), but that at high protein concentration (200 μg/mL) decreased (15%) due to aggregation comparing to that of the wild-type lysozyme (45%). However, the refolding yield of Ser-1 CaB lysozyme in the presence of 100 mM CaCl2 even at a protein concentration of 200 μg/mL was 80% and was higher than that of the wild-type lysozyme. From analysis of chemical shift changes of the cross peaks in the backbone region of total correlated spectroscopy (TOCSY) spectra of a decapeptide possessing the same calcium binding site as in Ser-1 CaB lysozyme in the presence of various concentrations of Ca2+, it was suggested that the dissociation constant of Ca2+-peptide complex was estimated to be 20-36 mM. Moreover, the solubility of the denatured Ser-1 CaB lysozyme in the presence of 100 mM CaCl2 was higher than that in the presence of 2 mM CaCl2 whereas the solubility of the denatured Ser-1 lysozyme in the presence of 100 mM CaCl2 was not higher than that in the presence of 2 mM CaCl2. Therefore, it was concluded that the reduced lysozyme possessing the Ca2+ binding site was efficiently folded in the presence of high concentration of Ca2+ (100 mM) even at high protein concentration due to depression of aggregation by the binding of Ca2+ to the polypeptide chain in Ser-1 CaB lysozyme.

本文言語英語
ページ(範囲)106-114
ページ数9
ジャーナルBiopolymers
64
2
DOI
出版ステータス出版済み - 7 5 2002

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Biomaterials
  • Organic Chemistry

フィンガープリント 「A metal binding in the polypeptide chain improves the folding efficiency of a denatured and reduced protein」の研究トピックを掘り下げます。これらがまとまってユニークなフィンガープリントを構成します。

引用スタイル