Engineering protein-carbohydrate interactions - lessons from natural and evolved carbohydrate binding modules

Sammanfattning: Protein-carbohydrate interactions are of central role in all living organism. In the studies presented in this thesis several strategies for engineering such interactions have been investigated. In the first study the binding affinity of a carbohydrate binding module (CBM) was improved a 100-fold through affinity maturation. Also, in that study it was discovered that all the selected, matured mutants carried a mutation in common (glutamic acid), which was shown to be responsible for the increased affinity. Furthermore, the binding interaction of two different CBM was analyzed with x-ray crystallography. One of the CBM can only bind xylans, while the other binds also beta-glucans and xyloglucan. The crystal structures revealed that the binding cleft of the multi-specific CBM is flexible, permitting reorientation of side-chains to avoid steric clashes. Also the multi-specific CBM harbored an important phenylalanine that due to its chemical composition has a pi-electron surface and can interact with hydrogen atoms on the different ligands it recognizes. The specific CBM on the other hand harbor a leucine in this position and can only interact with xylans. In the last study, the capacity of CBM for use as analytical tools in the characterization of the distribution pattern of cellulose derivatives was investigated. The study showed that some CBM are able to distinguish between cellulose derivatives with different substitution distribution. This is a potentially new application for CBM. In all, the thesis demonstrates strategies in which carbohydrate binding proteins can be generated, improved and utilized.