FRET Imaging of Diatoms Expressing a Biosilica-Localized Ribose Sensor
Future materials are envisioned to include bio-assembled, hybrid, three-dimensional nanosystems that incorporate functional proteins. Diatoms are amenable to genetic modification that enables localization of recombinant proteins in the biosilica cell wall. Our objective was to functionalize diatom biosilica with a reagent-less biosensor with FRET-based imaging capabilities for signaling. The design of the fusion protein conferring these 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 recombinant chimeric protein was first confirmed in E. coli-expressed proteins, prior to transformation of the diatom Thalassiosira pseudonana. Mass spectrometry of CRY showed 95% identity with the deduced amino acid sequence. CRY with and without an N-terminal Sil3 tag for biosilica targeting exhibited characteristic ribose-dependent changes in FRET, with similar dissociation constants of 123.3 {mu}M and 142.8 {mu}M, respectively. The addition of the silaffin tag for biosilica localization did not influence 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 biosilica 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.
- Research Organization:
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Environmental Molecular Sciences Lab. (EMSL)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- AC05-76RL01830
- OSTI ID:
- 1037518
- Report Number(s):
- PNNL-SA-84370; 44994; 400403309; TRN: US201207%%326
- Journal Information:
- PLoS One, Vol. 7, Issue 3
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
60 APPLIED LIFE SCIENCES
AFFINITY
AMINO ACID SEQUENCE
CELL WALL
CONFORMATIONAL CHANGES
DESIGN
DIATOMS
DISSOCIATION
FLUORESCENCE
FUNCTIONALS
GENETICS
MASS SPECTROSCOPY
MODIFICATIONS
PROTEINS
RIBOSE
SENSORS
TRANSFORMATIONS
VECTORS
Diatoms
FRET
Ribose receptor
biosilica immobilization
fluorescence
Environmental Molecular Sciences Laboratory