Structural studies of a cellulose binding protein by magic-angle apinning solid state nuclear magnetic resonance

In recent years, the search for biofuels has been a target in many studies. Cellulose, which is the most abundant carbohydrate polymer in nature, is a main candidate for this purpose. Cellulose degradation requires the synergistic involvement of numerous enzymes in association with a complex called the cellulosome. Carbohydrate binding modules (CBM) have an essential role in this degradation chain of reactions and CBM from family 3b (CBM3b) acts by creating scaffolding subunits for targeting the multi-enzyme complex to the crystalline cellulose. While the crystal structure of isolated CBM3b from Clostridium thermocellum exists, the exact form of binding to cellulose is not known since cellulose is insoluble, and cannot be co-crystalized with CBM3b.

As the first step towards characterization of the CBM-cellulose complex, we show results from 2D and 3D MAS ssNMR experiments on a fully and sparsely ¹³C and ¹⁵N labeled samples of isolated CBM3b. A partial assignment of the NMR chemical shifts was obtained, and the predicted secondary structure is in agreement with the reported X-ray structure. Furthermore, we used SDS-PAGE in order to quantify, but more importantly, to optimize the conditions which maximized the CBM-cellulose complex formation.

Various NMR experiments will be further implemented on the CBM-cellulose complex in order to obtain structural insight onto the calcium and cellulose binding sites.