Analyzing the weak dimerization of a cellulose binding module by sedimentation velocity experiments

Abstract

Cellulose binding modules (CBMs) are found widely in different proteins that act on cellulose. Because they allow a very easy way of binding recombinant proteins to cellulose, they have become widespread in many biotechnological applications involving cellulose. One commonly used variant is the CBMCipA from Clostridium thermocellum. Here we studied the dimerization of CBMCipA, because we were interested if its solution behavior could have an impact on its use in biotechnical applications. As the principal approach, we used sedimentation velocity analytical ultracentrifugation. To enhance our understanding of the possible interactions, we used molecular dynamics simulations. By analysis of the sedimentation velocity data using a discrete model genetic algorithm we found that the CBMCipA shows a weak dimerization interaction with a dissociation constant KD of about 87 M. As the KD of CBMCipA binding to cellulose is about 0.6 M, we conclude that the dimerization is unlikely to affect cellulose binding. However, at the high concentrations used in some applications of the CMBCipA, its dimerization is likely to have an effect on its solution behavior. The work shows that analytical ultracentrifugation is a very efficient tool to analyze this type of weak interactions. Moreover, we provide here a protocol for data analysis in the program Ultrascan for determining dissociation constants by sedimentation velocity experiments.