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Abstract
Future materials are envisioned to include bio-assembled, hybrid, three-dimensional nanosystems that incorporate functional proteins. Diatoms are amenable to genetic modification for localization of recombinant proteins in the biosilica cell wall. However, the full range of protein functionalities that can be accommodated by the modified porous biosilica has yet to be described. Our objective was to functionalize diatom biosilica with a reagent-less sensor dependent on ligand-binding and conformational change to drive FRET-based signaling capabilities. A fusion protein designed to confer such properties included a bacterial periplasmic ribose binding protein (R) flanked by CyPet (C) and YPet (Y), cyan and yellow fluorescent proteins that act as a FRET pair. The structure and function of the CRY recombinant chimeric protein was confirmed by expression in E. coli prior to transformation of the diatom Thalassiosira pseudonana. Mass spectrometry of the recombinant CRY showed 97% identity with the deduced amino acid sequence. CRY with and without an N-terminal Sil3 tag for biosilica localization exhibited characteristic ribose-dependent changes in FRET, with similar dissociation constants of 123.3 µM and 142.8 µM, respectively. The addition of the Sil3 tag did not alter the affinity of CRY for the ribose substrate. Subsequent transformation of T. pseudonana with a vector encoding Sil3-CRY resulted in fluorescence localization in the biosilica and changes in FRET in both living cells and isolated frustules in response to ribose. This work demonstrated that the nano-architecture of the genetically modified biosilica cell wall was able to support the functionality of the relatively complex Sil3-CyPet-RBP-YPet fusion protein with its requirement for ligand-binding and conformational change for FRET-signal generation.
Highlights
The construction of future three-dimensional materials with multiscale architectures is expected to include bio-assembly [1]
Construction and Characterization of the CRY Sensor The design of the CRY recombinant sensor was based on the FLIPrbs- F15A construct encoding ribose binding protein (RBP) flanked by enhanced cyan and yellow fluorescent proteins, ECFP and EYFP, respectively
We replaced the ECFP-EYFP Forster Resonance Energy Transfer (FRET) pair with sequences encoding CyPet and YPet fluorescent proteins [16] and cloned the resultant CyPet-RBP-YPet sequence into the pRSET vector for bacterial expression driven by a T7 promoter
Summary
The construction of future three-dimensional materials with multiscale architectures is expected to include bio-assembly [1]. Efforts to construct silica materials inspired by an understanding of diatom biology have included 1) silica condensation from silicic acid in vitro with the use of silaffins or silaffin-derived peptides [3,4,5,6,7] and 2) manipulation of living cells to add functional elements by metabolic insertion [8,9] or genetic modification of the cell wall structure [10,11] The latter cell-based approaches allow assembly under the ambient physical and chemical conditions inherent to diatom cell culture. Our objective was to test the ability of the diatom biosilica to serve as a scaffold for complex chimeric fusion proteins requiring large-scale motions associated with ligand-dependent conformational changes in order to function
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