Solid state NMR chemical shift assignment and conformational analysis of a cellulose binding protein facilitated by optimized glycerol enrichment.
Magic-angle spinning solid-state NMR has been applied to study CBM3b-Cbh9A (CBM3b), a cellulose binding module protein belonging to family 3b. It is a 146-residue protein having a unique nine-stranded β-sandwich fold, in which 35% of the structure is in a β-sheet conformation and the remainder of the protein is composed of loops and unstructured regions. Yet, the protein can be crystalized and it forms elongated needles. Close to complete chemical shift assignment of the protein was obtained by combining two- and three-dimensional experiments using a fully labeled sample and a glycerol-labeled sample. The use of an optimized protocol for glycerol-based sparse labeling reduces sample preparation costs and facilitates the assignment of the large number of aromatic signals in this protein. Conformational analysis shows good correlation between the NMR-predicted secondary structure and the reported X-ray crystal structure, in particular in the structured regions. Residues which show high B-factor values are situated mainly in unstructured regions, and are missing in our spectra indicating conformational flexibility rather than heterogeneity. Interestingly, long-range contacts, which could be clearly detected for tyrosine residues, could not be observed for aromatic phenylalanine residues pointing into the hydrophobic core, suggesting possible high ring mobility. These studies will allow us to further investigate the cellulose-bound form of CBM proteins.
Raymond and Beverly Sackler Faculty of Exact Sciences, School of Chemistry, Ramat Aviv, 69978, Tel Aviv, Israel.
Hadar
H
Ivanir
Amir
A
Goldbourt
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry*; D002482:Cellulose / Q000737:chemistry*; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D005990:Glycerol / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D017433:Protein Structure, Secondary
D002352:Carrier Proteins; D002482:Cellulose; D005990:Glycerol