Unassisted refolding of urea‐denatured arginine kinase from shrimp Feneropenaeus chinensis: Evidence for two equilibrium intermediates in the refolding pathway

Protein Science - Tập 13 Số 7 - Trang 1892-1901 - 2004
Ji-Cheng Pan1, Zhenhang Yu2, Xiaoyang Su2, Yingxin Sun3, Xue-Ming Rao4, Hai‐Meng Zhou5
1Department of Biological Science and Biotechnology, School of Life Science and Engineering, Tsinghua University, Bejing, China.
2Department of Biological Science and Biotechnology, Tsinghua University, Beijing 100084, China
3Department of Engineering and Life Science, Harbin Institute of Technology, Harbin 100051, Heilongjiang, China
4College of Agronomy Henan Agricultural University Zhengzhou Henan 450002 China
5Protein Science Laboratory of the Ministry of Education, School of Life Science and Engineering, Tsinghua University, Beijing 100084, China

Tóm tắt

AbstractThe refolding process and the equilibrium intermediates of urea‐denatured arginine kinase (AK) were investigated by 1‐anilino‐8‐naphthalenesulfonate (ANS) intrinsic fluorescence, far‐UV circular dichroism (CD), size‐exclusion chromatography (SEC), and enzymatic activity. In dilute denaturant, two equilibrium refolding intermediates (I and N′) were discovered, and a refolding scheme of urea‐denatured AK was proposed. During the refolding of urea‐denatured AK, the fluorescence intensity increased remarkably, accompanied by a significant blue shift of the emission maximum and a pronounced increase in molar ellipticity of CD at 222 nm. The first folding intermediate (I) was inactive in urea solution ranging between 2.4 and 3.0 M. The second (N′) existed between a 0.4‐ and 0.8‐M urea solution, with slightly increased activity. Neither the blue shift emission maximum nor the molar ellipticity of CD at 222 nm showed significant changes in these two regions. The two intermediates were characterized by monitoring the ANS binding ability in various residual urea solutions, and two peaks of the emission intensity were observed in urea solutions of 0.6 and 2.8 M, respectively. The SEC results indicated that a distribution coefficient (KD) platform existed in urea solutions ranging between 2.4 and 3.0 M urea, suggesting that there was a similarly apparent protein profile and size in the urea solution region. The refolding kinetics showed that the urea‐denatured AK was in two‐phase refolding. Proline isomerization occurred in the unfolding process of AK, which blocked the slow phase of refolding. These results suggested that the refolding process of urea‐denatured AK contained at the least two equilibrium refolding intermediates.

Từ khóa


Tài liệu tham khảo

10.1126/science.181.4096.223

10.1016/S0167-4838(98)00282-9

10.1021/bi9929103

10.1021/bi00693a026

10.1110/ps.8.11.2258

10.1002/(SICI)1097-0134(20000515)39:3<216::AID-PROT40>3.0.CO;2-#

10.1002/pro.5560071217

10.1126/science.70.1816.381-a

10.1016/S0022-2860(96)09289-7

10.1016/S0006-3495(98)77590-3

10.1006/abbi.1996.0051

10.1006/abbi.1997.0243

10.1038/381341a0

10.1021/bi00429a004

10.1073/pnas.87.2.573

10.1021/bi002553s

10.1021/bi00033a005

10.1002/prot.340060202

10.1096/fasebj.10.1.8566530

10.1093/oxfordjournals.jbchem.a021295

10.1016/0167-4838(90)90211-W

10.1006/abbi.2001.2529

10.1042/bj2770207

10.1073/pnas.022387699

10.1006/jmbi.1999.2769

10.1007/BF01267958

10.1007/BF00248050

10.1016/S0065-3233(08)60546-X

10.1016/S0014-5793(98)01355-6

10.1021/bi00289a013

10.1110/ps.47101

Stryer L., 1965, The interaction of a naphthalene dye with apomyoglobin and apohemoglobin. A fluorescent probe of non‐polar binding sites, J. Mol. Biol., 245, 525

10.1042/bj3280301

10.1042/bj3400671

10.1016/S1357-2725(02)00050-X

10.1042/bj0940536

Watts D.C., 1975, Evolution of phosphagens along the chordate line, Symp. Zool. Soc. Lond., 36, 105

10.1042/bj3081025

10.1016/0005-2728(92)90096-K

10.1016/S0167-4838(97)00026-5

10.1107/S0907444902014683

10.1038/nsb995

10.1042/bj2910103

10.1073/pnas.95.15.8449

10.1016/S0167-4838(00)00244-2

Thông tin
Thông tin xuất bản

Protein Science

Tập 13 Số 7

1892-1901

Thông tin tác giả