Investigating amino acid contribution to mOrange and mStrawberry spectra

  • Kyle Poffenroth Mount Royal University
  • Evans Eshrew Mount Royal University
  • Jennifer Flood Mount Royal University
  • Shae Konschuh Mount Royal University
  • Stephanie Rothwell Mount Royal University
  • Maryam Yaqoob Mount Royal University
  • David Bird* Mount Royal University

Abstract

Fluorescent proteins (FP) contain a unique fluorochrome, usually composed of 3 amino acids, which can absorb and emit light. FPs are widely used in cell biology by fusing them with target proteins and imaging the chimeric protein using fluorescence microscopy. Thus, FPs allow biologists to monitor cellular processes in vivo, for example, visualizing cytoskeletal components or track moving proteins and/or organelles. Directed evolution of fluorescence proteins has created a variety of colors, allowing multiple cellular components to be distinguished simultaneously within the same cell. While mOrange has a lower wavelength emission compared to dsRED, mStrawberry has a higher wavelength. There are seven amino acid differences between these FP variants, some of which are believed to be directly responsible for the change in emission, while the contribution of others is less clear. This project investigates contributions two of these residues found at position 62 and 213. Using site-directed mutagenesis was used to change glutamine at position 213 to leucine (Q213L) and serine at position 62 to threonine (S62T). Residue 62 lies within the chromophore of mOrange, thus changing the amino acid here will directly alter the chromophore. We predict that Q213L will alter the microenvironment near the fluorochrome due to its proximity to it (within 5 Å). We expect the net result to be a red-shift of mOrange towards mStrawberry. Research in this area can provide new information about how these proteins produce chromophores and the effects of the microenvironment within the protein on the fluorochrome.


* Indicates faculty mentor.

Published
2017-04-27
Section
Poster Abstracts - Biological Science