Imaging Structurally Heterogeneous Protein Complexes in Diatoms transformed with mesoporous biosilica targeted proteins
EMSL Project ID
48780
Abstract
Our long-term goal is to monitor location and functionally-relevant structural transitions of proteins stabilized by in vivo sol-gel condensation of protein-silica composite structures. Such measurements are anticipated to permit development of predictive rules for the construction of optimal biosilica-based biomaterials, which include catalysis platforms for biofuels. The biarsenical fluorescent probes AsCy3/AsCy3e, which bind the tetracysteine tag AsCy3Tag encoded in fusion proteins, have been useful in localizing biosilica-targeted recombinant proteins in both nascent and mature biosilica of transformed diatoms. An improved tag sequence (Cy3+6Tag) that permits higher affinity and enhanced fluorescence detection at the single peptide level was recently reported, and we have extended use of this tag to detection of recombinant proteins in biosilica of transformed diatoms using epifluorescence microscopy. A better understanding of the localization of the tagged proteins in both biosilica and non-biosilica structures can be gained through high-resolution imaging of labeled cells and isolated biosilica. The specific aim of this Rapid Access proposal is to compare the original Cy3Tag and the new Cy3+6Tag in resolving localization of labeled proteins using Super Resolution Structured Illumination Microscopy (3D-SIM). The results of 3D-SIM imaging are needed to complement and strengthen existing comparisons using lower resolution imaging approaches. The findings, which are planned for immediate submission for publication and subsequent presentation at a major scientific conference and program review, would extend the recently reported proof of principle in vitro biochemical study to tagged live cells and isolated biosilica frustules.
Project Details
Project type
Limited Scope
Start Date
2015-03-16
End Date
2015-05-16
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members