Terminal spin labeling of xylotriose strongly affects interactions in the active site of xylanase BcX

被引：0

作者：

Saberi, Mahin ^{[1
]}

Dekkers, Rene ^{[1
,2
]}

Passerini, Leonardo ^{[3
]}

Huber, Martina ^{[3
]}

Overhand, Mark ^{[2
]}

Ubbink, Marcellus ^{[1
]}

机构：

[1] Leiden Univ, Leiden Inst Chem, Dept Macromol Biochem, Einsteinweg 55, NL-2333 CC Leiden, Netherlands

[2] Leiden Univ, Leiden Inst Chem, Dept Bioorgan Synth, Einsteinweg 55, NL-2333 CC Leiden, Netherlands

[3] Leiden Univ, Dept Phys, Huygens Kamerlingh Onnes Lab, Niels Bohrweg 2, NL-2333 CA Leiden, Netherlands

来源：

JOURNAL OF BIOMOLECULAR NMR | 2025年

关键词：

Xylanase; Spin label; Transient interactions; NMR spectroscopy; Paramagnetic relaxation enhancement; TEMPO; PROTEIN-LIGAND INTERACTIONS; MOLECULAR-DYNAMICS; BINDING; CONFORMATION; SUBSITES; COMPLEX; DOMAIN; STATES;

D O I：

10.1007/s10858-025-00459-w

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：

Paramagnetic probes provide long-range distance information and report on minor conformations of biomacromolecules. However, it is important to realize that any probe can affect the system of interest. Here, we report on the effects of attaching a small nitroxide spin label [TEMPO, (2,2,6,6-tetramethylpiperidin-1-yl)oxyl] to xylotriose, a substrate of the enzyme xylanase from Bacillus circulans (BcX). BcX has a long and narrow active site cleft accommodating six xylose units and a secondary binding site on its surface. The aim of the study was to probe the interactions of the substrate with the enzyme using paramagnetic relaxation enhancements (PREs). Binding of the substrate to the surface exposed secondary binding site resulted in strong and localized PREs, indicative of well-defined binding. The xylotriose with diamagnetic control tag was still able to bind the active site cleft, though the rate of exchange was reduced relative to that of untagged xylotriose. The substrate with the paramagnetic TEMPO was not able to bind inside the active site cleft. Also, additional interactions on another surface location showed differences between the paramagnetic substrate and the diamagnetic control, despite the minimal chemical differences between TEMPO modified xylotriose and its reduced, diamagnetic counterpart. Our findings underscore the sensitivity of BcX substrate binding to minor substrate modifications. This study serves as a reminder that any probe, including the attachment of a small paramagnetic group, can affect the behavior of the system under investigation. Even the chemical difference between a paramagnetic tag and its diamagnetic control can result in differences in the molecular interactions.

引用

页码：99 / 113

页数：15

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