通过多个位点的顺磁性镧系标签的伪接触位移定位金属-β-内酰胺酶IMP-1中色氨酸侧链的单个原子。

Q3 Physics and Astronomy
Magnetic resonance (Gottingen, Germany) Pub Date : 2022-01-04 eCollection Date: 2022-01-01 DOI:10.5194/mr-3-1-2022
Henry W Orton, Iresha D Herath, Ansis Maleckis, Shereen Jabar, Monika Szabo, Bim Graham, Colum Breen, Lydia Topping, Stephen J Butler, Gottfried Otting
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引用次数: 0

摘要

金属-β-内酰胺酶IMP-1在活性位点附近有一个柔性环,在单晶结构中呈现不同的构象,这可能有助于底物结合和酶活性。为了探测这个环的位置,我们用7-13C-吲哚标记IMP-1的色氨酸残基,并在三个不同的位点用镧系元素标签标记蛋白质。通过测量主链酰胺质子的伪接触位移(PCSs)来确定磁化率各向异性(Δχ)张量。随后使用Δχ张量来确定蛋白质中色氨酸侧链的原子坐标。PCS足以以高精度确定Trp28的位置,Trp28位于我们实验靶向的活性位点环中。其平均原子坐标对抑制剂卡托普利的反应几乎没有显著变化。研究发现,通过仅包括每个标签和标签位点的单个顺磁性镧系元素离子的PCS,可以更准确地定义定位空间。这种效应归因于PCS等表面倾向于相交的浅角度,如果由除顺磁性镧系离子外相同的标签和标签位点产生的话。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Localising individual atoms of tryptophan side chains in the metallo-<i>β</i>-lactamase IMP-1 by pseudocontact shifts from paramagnetic lanthanoid tags at multiple sites.

Localising individual atoms of tryptophan side chains in the metallo-<i>β</i>-lactamase IMP-1 by pseudocontact shifts from paramagnetic lanthanoid tags at multiple sites.

Localising individual atoms of tryptophan side chains in the metallo-<i>β</i>-lactamase IMP-1 by pseudocontact shifts from paramagnetic lanthanoid tags at multiple sites.

Localising individual atoms of tryptophan side chains in the metallo-β-lactamase IMP-1 by pseudocontact shifts from paramagnetic lanthanoid tags at multiple sites.

The metallo-β-lactamase IMP-1 features a flexible loop near the active site that assumes different conformations in single crystal structures, which may assist in substrate binding and enzymatic activity. To probe the position of this loop, we labelled the tryptophan residues of IMP-1 with 7-13C-indole and the protein with lanthanoid tags at three different sites. The magnetic susceptibility anisotropy (Δχ) tensors were determined by measuring pseudocontact shifts (PCSs) of backbone amide protons. The Δχ tensors were subsequently used to identify the atomic coordinates of the tryptophan side chains in the protein. The PCSs were sufficient to determine the location of Trp28, which is in the active site loop targeted by our experiments, with high accuracy. Its average atomic coordinates showed barely significant changes in response to the inhibitor captopril. It was found that localisation spaces could be defined with better accuracy by including only the PCSs of a single paramagnetic lanthanoid ion for each tag and tagging site. The effect was attributed to the shallow angle with which PCS isosurfaces tend to intersect if generated by tags and tagging sites that are identical except for the paramagnetic lanthanoid ion.

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来源期刊
CiteScore
4.50
自引率
0.00%
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14 weeks
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