Generation of a fluorescent protein-derived biosensor based on alternate frame folding

• Author / Creator

  Saini, Ritesh K.

• To develop a single fluorescent protein (FP)-based scaffold that could potentially serve as a universal platform for biosensor creation, we have introduced the tandem alternate frame switching strategy. We genetically inserted a non-identical copy of 7th β-strand in Aequorea victoria greenFP(avGFP) to create protein conformational switch. By creating a series of variants, we demonstrated that equilibrium between conformations can be manipulated by substitution, deletion and insertion of residues. The chromophore absorption spectra unique to each conformation allowed the determination of bound equilibrium through independent component analysis. To verify the potential of this strategy, we strategically created calcium-ion (Ca2+) and cyclic adenosine-monophosphate (cAMP) biosensors and evaluated the change in the fluorescence spectra corresponding to binding of the analyte. We performed preliminary study to explore switch generation in cpmCherry. We anticipate that this rational strategy for construction of genetically-encoded indicators could be applicable to a variety of FPs and sensing motifs.

• Subjects / Keywords

  • AvGFP
  • Alternate frame folding
  • Tandem alternate frame switching

• Graduation date

  Fall 2012

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/R3057D01P

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.