STRUCTURAL STUDIES OF A CELLULOSE BINDING MODULE PROTEIN BY MAGIC ANGLE SPINNING SOLID STATE NMR

In recent years, the search for biofuels has been the target of many scientific and industrial studies. Cellulose, which is the most abundant carbohydrate polymer in nature, is one of the main candidates for mass biofuel production. One of the routes for cellulose degradation requires the synergistic involvement of several enzymes in association with a complex called the cellulosome. Carbohydrate binding modules (CBMs) have an essential role in this degradation process; CBMs bind to the cellulose surface thereby bringing the attached enzymes to a close proximity to the carbohydrate polymer allowing its decomposition.

The crystal structures of family 3b CBM from Clostridium thermocellum (ctCBM3b) and of one of its mutants (N105W) that binds very strongly to cellulose have been determined by X-ray crystallography (pdb 2ylk, 3zqx) and it has been assumed that the three aromatic residues of the mutant are involved in stacking interactions with the glucose rings of the cellulose chains, as this motif is preserved in most proteins of the CBM family. To date, there are no structures of cellulose-bound proteins and therefore no structural evidence for such binding.

Using Magic-angle spinning (MAS) solid-state NMR, we aim to obtain structural insights into the CBM3b^N105W protein and in particular to allocate and characterize its cellulose binding site and interactions. In this study, we show studies of the free protein obtained from performing 2D and 3D MAS ssNMR experiments on the fully ¹³C and ¹⁵N labeled protein.