Expression, purification and the 1.8 Å resolution crystal structure of human neuron specific enolase

被引:50
作者
Chai, GQ
Brewer, JM
Lovelace, LL
Aoki, T
Minor, W
Lebioda, L [1 ]
机构
[1] Univ S Carolina, Dept Chem & Biochem, Columbia, SC 29208 USA
[2] Univ Georgia, Dept Biochem & Mol Biol, Athens, GA 30602 USA
[3] Hokkaido Univ, Dept Biochem Hlth Sci, Sapporo, Hokkaido 060, Japan
[4] Univ Virginia, Dept Mol Physiol & Biol Phys, Charlottesville, VA 22901 USA
基金
美国国家科学基金会;
关键词
enolase; neurons; isozymes; surface charges; crystal structure;
D O I
10.1016/j.jmb.2004.05.068
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Human neuron-specific enolase (NSE) or isozyme gamma has been expressed with a C-terminal His-tag in Escherichia coli. The enzyme has been purified, crystallized and its crystal structure determined. In the crystals the enzyme forms the asymmetric complex (NSEMg2SO4)-Mg-.-S-./(NSEMgCl)-Mg-.-Cl-., where "/" separates the dimer subunits. The subunit that contains the sulfate (or phosphate) ion and two magnesium ions is in the closed conformation observed in enolase complexes with the substrate or its analogues; the other subunit is in the open conformation observed in enolase subunits without bound substrate or analogues. This indicates negative cooperativity for ligand binding between subunits. Electrostatic charge differences between isozymes alpha and gamma, - 19 at physiological pH, are concentrated in the regions of the molecular surface that are negatively charged in alpha, i.e. surface areas negatively charged in alpha are more negatively charged in gamma, while areas that are neutral or positively charged tend to be charge-conserved. (C) 2004 Elsevier Ltd. All rights reserved.
引用
收藏
页码:1015 / 1021
页数:7
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