Analyzing the weak dimerization of a cellulose binding module by analytical ultracentrifugation

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 CBM_CipA from Clostridium thermocellum. Here we studied the oligomerization behavior of CBM_CipA, as such solution association may have an impact on its use. As the principal approach, we used sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation. To enhance our understanding of the possible interactions, we used molecular dynamics simulations. By analysis of the sedimentation velocity data by a discrete model genetic algorithm and by building a binding isotherm based on weight average sedimentation coefficient and by global fitting of sedimentation equilibrium data we found that the CBM_CipA shows a weak dimerization interaction with a dissociation constant K_D of 90 ± 30 μM. As the K_D of CBM_CipA binding to cellulose is below 1 μM, we conclude that the dimerization is unlikely to affect cellulose binding. However, at high concentrations used in some applications of the CBM_CipA, its dimerization is likely to have a marked effect on its solution behavior.