Expression and Spectroscopic Analysis of Soluble Nicotinic Acetylcholine Receptor Fragments Derived from the Extracellular Domain of the 
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- 作者：Marianne A. Grant ; Lisa N. Gentile ; Qing-Luo Shi ; Maria Pellegrini ; and Edward Hawrot
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：August 17, 1999
- 年：1999
- 卷：38
- 期：33
- 页码：10730 - 10742
- 全文大小：247K
- 年卷期：v.38,no.33(August 17, 1999)
- ISSN：1520-4995
文摘
To facilitate structural studies of the ligand binding region from the nicotinic acetylcholinereceptor (nAChR), we have developed methods for the high-level expression and purification of an importantfunctional portion of the N-terminal extracellular domain (ECD) of the 
-subunit. Two soluble receptorfragments comprising residues 143-210 of the Torpedo californica -subunit were expressed in E. coli:T68His6, which contains a histidine tag, and T68M1, which includes the first transmembrane region,M1, of the -subunit. Both proteins demonstrate saturable, high-affinity -bungarotoxin (Bgtx) bindingwith an apparent equilibrium KD (3 nM) that is comparable to the affinities reported for preparationscomprising the entire -subunit ECD. These results demonstrate that the ECD determinants required forBgtx recognition of the -subunit are entirely specified by residues 143-210. The binding of small ligandswas demonstrated in competition assays with 125I-Bgtx yielding KI values of 58 and 105 
M ford-tubocurarine and nicotine, respectively. Circular dichroism (CD) analysis of monomeric T68His6 proteinrevealed considerable secondary structure. Furthermore, a cooperative, two-state folding transition wasobserved upon urea denaturation. To circumvent concentration-dependent aggregation of the T68His6protein at the millimolar concentrations needed for NMR study, we utilized the M1 transmembrane domainto anchor the recombinant receptor fragment onto membrane-mimicking micelles. Monodispersed preparations of T68M1 in dodecylphosphocholine micelles demonstrate high-affinity Bgtx binding and considerable secondary structure by CD. The structural features revealed in the CD profile appear to undergo acooperative, two-state folding transition upon thermal denaturation. Initial NMR studies suggest that micellarpreparations of the T68M1 fragment are amenable to further high-resolution heteronuclear NMR analysis.
-subunit. Two soluble receptorfragments comprising residues 143-210 of the Torpedo californica -subunit were expressed in E. coli:T68His6, which contains a histidine tag, and T68M1, which includes the first transmembrane region,M1, of the -subunit. Both proteins demonstrate saturable, high-affinity -bungarotoxin (Bgtx) bindingwith an apparent equilibrium KD (3 nM) that is comparable to the affinities reported for preparationscomprising the entire -subunit ECD. These results demonstrate that the ECD determinants required forBgtx recognition of the -subunit are entirely specified by residues 143-210. The binding of small ligandswas demonstrated in competition assays with 125I-Bgtx yielding KI values of 58 and 105 M ford-tubocurarine and nicotine, respectively. Circular dichroism (CD) analysis of monomeric T68His6 proteinrevealed considerable secondary structure. Furthermore, a cooperative, two-state folding transition wasobserved upon urea denaturation. To circumvent concentration-dependent aggregation of the T68His6protein at the millimolar concentrations needed for NMR study, we utilized the M1 transmembrane domainto anchor the recombinant receptor fragment onto membrane-mimicking micelles. Monodispersed preparations of T68M1 in dodecylphosphocholine micelles demonstrate high-affinity Bgtx binding and considerable secondary structure by CD. The structural features revealed in the CD profile appear to undergo acooperative, two-state folding transition upon thermal denaturation. Initial NMR studies suggest that micellarpreparations of the T68M1 fragment are amenable to further high-resolution heteronuclear NMR analysis.