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CELLULOSIC biomass is recycled by a variety of microorganisms occupying different habitats 1 . Studies of their cellulase systems have included the purification of enzyme components, the determination of their enzymological properties 2 and the cloning and characterization of their structural genes 3 . Sequence analysis of more than 70 cellulases permits grouping into seven families corresponding to distinct structural types 4,5 . The three-dimensional structure of the catalytic core of cellobiohydrolase CBHII from the fungus Trichoderma reesei has been reported 6 . Here we show that endoglucanase CelD from Clostridium thermocellum , which is representative of a different family of cellulose-degrading enzymes consisting of at least 11 bacterial, fungal and plant endoglucanases 5,7 , has a globular structure, with an amino-terminal immunoglobulin-like domain tightly packed against a larger catalytic domain. The latter shows a novel protein fold, shaped like an α-barrel of 12 helices connected by loops that form the active site. The structure of a complex CelD with a substrate analogue suggests a mechanism for substrate hydrolysis.

Solid state NMR sample preparation and resonance assignments of the U-[^sup 13^C,^sup 15^N] 2×10.4 kDa dimeric form of the regulatory protein Crh in microcrystalline, PEG precipitated form are presented. Intra- and interresidue correlations using dipolar polarization transfer methods led to nearly complete sequential assignments of the protein, and to 88% of all ^sup 15^N, ^sup 13^C chemical shifts. For several residues, the resonance assignments differ significantly from those reported for the monomeric form analyzed by solution state NMR. Dihedral angles obtained from a TALOS-based statistical analysis suggest that the microcrystalline arrangement of Crh must be similar to the domain-swapped dimeric structure of a single crystal form recently solved using X-ray crystallography. For a limited number of protein residues, a remarkable doubling of the observed NMR resonances is observed indicative of local static or dynamic conformational disorder. Our study reports resonance assignments for the largest protein investigated by solid state NMR so far and describes the conformational dimeric variant of Crh with previously unknown chemical shifts.[PUBLICATION ABSTRACT]

We report site-resolved observation of hydrogen exchange in the micro-crystalline protein Crh. Our approach is based on the use of proton T2' -selective 1H-13C-13C correlation spectra for site-specific assignments of carbons nearby labile protein protons. We compare the proton T2' selective scheme to frequency selective water observation in deuterated proteins, and discuss the impacts of deuteration on 13C linewidths in Crh. We observe that in micro-crystalline proteins, solvent accessible hydroxyl and amino protons show comparable exchange rates with water protons as for proteins in solution, and that structural constraints, such as hydrogen bonding or solvent accessibility, more significantly reduce exchange rates.

Solid state NMR sample preparation and resonance assignments of the U-[13C,15N] 2x10.4 kDa dimeric form of the regulatory protein Crh in microcrystalline, PEG precipitated form are presented. Intra- and interresidue correlations using dipolar polarization transfer methods led to nearly complete sequential assignments of the protein, and to 88% of all 15N, 13C chemical shifts. For several residues, the resonance assignments differ significantly from those reported for the monomeric form analyzed by solution state NMR. Dihedral angles obtained from a TALOS-based statistical analysis suggest that the microcrystalline arrangement of Crh must be similar to the domain-swapped dimeric structure of a single crystal form recently solved using X-ray crystallography. For a limited number of protein residues, a remarkable doubling of the observed NMR resonances is observed indicative of local static or dynamic conformational disorder. Our study reports resonance assignments for the largest protein investigated by solid state NMR so far and describes the conformational dimeric variant of Crh with previously unknown chemical shifts.