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Protein Disorder: Conformational Distribution of the Flexible Linker in a Chimeric Double Cellulase

Ingemar von Ossowski ^*, Julian T. Eaton , Mirjam Czjzek , Stephen J. Perkins , Torben P. Frandsen ^*, Martin Schülein ^* , Pierre Panine , Bernard Henrissat  and Veronique Receveur-Bréchot 

^* Novozymes A/S, Bagsvaerd, Denmark; Department of Biochemistry and Molecular Biology, University College London, London, United Kingdom; Architecture et Fonction des Macromolécules Biologiques, UMR 6098, Centre National de la Recherche Scientifique and Universités d'Aix-Marseille I and II, Marseille, France; and European Synchrotron Radiation Facility, Grenoble, France

Correspondence: Address reprint requests to Véronique Receveur-Bréchot. E-mail: receveur{at}afmb.cnrs-mrs.fr.

The structural properties of the linker peptide connecting the cellulose-binding module to the catalytic module in bimodular cellulases have been investigated by small-angle x-ray scattering. Since the linker and the cellulose-binding module are relatively small and cannot be readily detected separately, the conformation of the linker was studied by means of an artificial fusion protein, Cel6BA, in which an 88-residue linker connects the large catalytic modules of the cellulases Cel6A and Cel6B from Humicola insolens. Our data showed that Cel6BA is very elongated with a maximum dimension of 178 Å, but could not be described by a single conformation. Modeling of a series of Cel6BA conformers with interdomain separations ranging between 10 Å and 130 Å showed that good Guinier and P(r) profile fits were obtained by a weighted average of the scattering curves of all the models where the linker follows a nonrandom distribution, with a preference for the more compact conformers. These structural properties are likely to be essential for the function of the linker as a molecular spring between the two functional modules. Small-angle x-ray scattering therefore provides a unique tool to quantitatively analyze the conformational disorder typical of proteins described as natively unfolded.