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

IF 1.3 3区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY Journal of Biomolecular NMR Pub Date : 2025-03-12 DOI:10.1007/s10858-025-00459-w
Mahin Saberi, René Dekkers, Leonardo Passerini, Martina Huber, Mark Overhand, Marcellus Ubbink
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Abstract

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.

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来源期刊
Journal of Biomolecular NMR
Journal of Biomolecular NMR 生物-光谱学
CiteScore
6.00
自引率
3.70%
发文量
19
审稿时长
6-12 weeks
期刊介绍: The Journal of Biomolecular NMR provides a forum for publishing research on technical developments and innovative applications of nuclear magnetic resonance spectroscopy for the study of structure and dynamic properties of biopolymers in solution, liquid crystals, solids and mixed environments, e.g., attached to membranes. This may include: Three-dimensional structure determination of biological macromolecules (polypeptides/proteins, DNA, RNA, oligosaccharides) by NMR. New NMR techniques for studies of biological macromolecules. Novel approaches to computer-aided automated analysis of multidimensional NMR spectra. Computational methods for the structural interpretation of NMR data, including structure refinement. Comparisons of structures determined by NMR with those obtained by other methods, e.g. by diffraction techniques with protein single crystals. New techniques of sample preparation for NMR experiments (biosynthetic and chemical methods for isotope labeling, preparation of nutrients for biosynthetic isotope labeling, etc.). An NMR characterization of the products must be included.
期刊最新文献
Terminal spin labeling of xylotriose strongly affects interactions in the active site of xylanase BcX. Improvement in protein HSQC spectra from addition of betaine. Exploring the biochemical landscape of bacterial medium with pyruvate as the exclusive carbon source for NMR studies. Counterintuitive method improves yields of isotopically labelled proteins expressed in flask-cultured Escherichia coli. Local structure propensities in disordered proteins from cross-correlated NMR spin relaxation.
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