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

Abstract

12Cellulose binding modules (CBMs) are found widely in different proteins that act on cellulose. 13Because they allow a very easy way of binding recombinant proteins to cellulose, they have 14 become widespread in many biotechnological applications involving cellulose. One commonly 15 used variant is the CBMCipA from Clostridium thermocellum. Here we studied the dimerization of 16CBMCipA, because we were interested if its solution behavior could have an impact on its use in 17 biotechnical applications. As the principal approach, we used sedimentation velocity analytical 18 ultracentrifugation. To enhance our understanding of the possible interactions, we used molecular 19 dynamics simulations. By analysis of the sedimentation velocity data using a discrete model 20 genetic algorithm we found that the CBMCipA shows a weak dimerization interaction with a 21 dissociation constant KD of about 87 µM. As the KD of CBMCipA binding to cellulose is about 0.6 22 µM, we conclude that the dimerization is unlikely to affect cellulose binding. However, at the high 23 concentrations used in some applications of the CMBCipA, its dimerization is likely to have an 24 effect on its solution behavior. The work shows that analytical ultracentrifugation is a very efficient 25 tool to analyze this type of weak interactions. Moreover, we provide here a protocol for data 26 analysis in the program Ultrascan for determining dissociation constants by sedimentation velocity 27 experiments. 28