Design of Bioresponsive Interfaces using Conformational Transitions of Calmodulin

Abstract

Some proteins undergo major conformational changes upon ligand binding, such as calmodulin (CaM). When CaM binds trifluoperazine (TFP), there is a marked transition of CaM from an open to a collapsed conformation, as can be seen in the distance distribution function obtained by small-angle X-ray scattering (SAXS). This functionality is transferred to interfacial structures in this project. Aqueous-solid interfaces are modified by covalent binding of PEG chains to the solid phase, which are cross-linked with a CaM mutant. The main achievements of this project are the controlled build-up of such interfacial layers and the characterization of the CaM functionality in these layers. Using X-ray reflectometry, we have observed that a PEG-CaM layer is growing in thickness upon rinsing with a TFP/Ca2+ solution and decreasing in thickness upon rinsing with an EGTA solution, which binds Ca2+ from CaM. Furthermore, pressure has been applied to probe the strength of interactions between CaM and TFP in solution and in the PEG-CaM layer, which are expected to be mainly electrostatic in nature. By measuring the fluorescence resonance energy transfer (FRET), we see a pressure-induced transition of collapsed CaM-TFP to open CaM-TFP free in solution. However, this transition cannot be observed in the PEG-CaM-TFP layer suggesting a stabilization of the collapsed state. Overall, novel biochemically responsive interfacial structures are formed that can be switched reversibly under ambient conditions, in contrast to well-known pH or temperature sensitive surface modifications.