Characterization of protein immobilization in a calmodulin hydragel

Brenda Marie Beech

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 was 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 an approximately 35° rigid body domain reorientation upon binding of maltodextrins that can be detected by solution nuclear magnetic resolution (NMR) spectroscopy. We hypothesized that through the study of the interactions of MBP with the CaM hydrogel, 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, M13, which resulted in the immobilization of MBP-M13 to a hydrogel composed of cys-mutated CaM cross-linked to poly(ethylene glycol) diacrylate (PEGDA). Immobilization, function, and stability of MBP-M13 were confirmed by fluorescence and NMR spectroscopies. We found that the amount of mobility MBP retained upon immobilization was dependent on the mesh network size of the hydrogel. The mesh network was affected by pressure, swollen state, and PEG length used in the hydrogel system. This hydrogel system appears to be insensitive to pH changes and to the gel formation process. We believe that our CaM-PEG hydrogel has the potential to functionally immobilize a broad range of proteins and seems like a model system for investigating the optimal material properties of general immobilization systems. Through our study of MBP immobilized in a CaM-PEG hydrogel, we conclude it is important for immobilization materials to be nonreactive scaffolds that specifically orient the protein within adequately sized mesh networks.

Characterization of protein immobilization in a calmodulin hydragel

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