Physical Properties of a Calmodulin Hydrogel as a Scaffold for Protein Immobilization

Current approaches in the study of protein function following immobilization on solid supports do not address how the properties of the support material affect protein structure and dynamics. In order to more efficiently create appropriate immobilization systems, general principles regarding the interaction between the material properties and broad classes of proteins need to be understood. Our goal is to gain some insight as to what material properties are optimal in the functional immobilization of proteins through the study of maltose binding protein (MBP) immobilized in a calmodulin (CaM) hydrogel. Maltose binding protein undergoes a ∼35° rigid body domain reorientation upon binding of sugar that can be detected by solution NMR. We hypothesize that through the study of the interactions of MBP with the CaM hydrogel (through a range of pHs) we can form some general principles regarding material:protein interfaces that will be applicable in the functional immobilization of broad classes of proteins. In this study we tagged MBP with a peptide target of calmodulin which immobilized MBP to a micro-sized spherical hydrogel composed of Cys-mutated CaM cross-linked to poly(ethylene glycol) diacrylate (PEGDA). Immobilization of MBP was confirmed by fluorescence and NuPAGE protein gels, and gel optimization for maximum immobilization is underway. Various NMR experiments have been and will be done in order to confirm function and to analyze the interactions between the immobilized protein and the hydrogel.