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

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.