Roger Tsien died a few days ago, at the relatively young age of 64. He was a UCSD scientist, a Nobel laureate and he was one of the first to see the significance and usefulness of GFP.
I’ve never met him. But, I guess, this blogs owes him its existence.
I don’t want to discuss his body of work, his achievements, or awards he won (e.g. the Nobel award). Many wrote nice things about him, such as here, here or here and all over the internet, with nice pictures of fluorescent proteins used in research.
I thought it will be nice to look back at his first GFP paper.
His goal in this paper was to investigate the formation of the fluorophore of GFP. Specifically, he asked:
“What is the mechanism of fluorophore formation? How does fluorescence relate to protein structure? Can its fluorescence properties be tailored and improved-in particular, to provide a second distinguishable color for comparison of independent proteins and gene expression events?”
Already here he looked to utilize GFP – to improve it, to change it, so it can be useful for fluorescent studies in biology.
He used random mutagenesis of the GFP cDNA to screen for mutants with altered brightness and emission. A simple yet powerful method, still used today, to find new FPs with exciting and useful properties.
Here is an ex/em spectral analysis of some of the mutants:
One mutant, I167T, proved to be almost twice as bright as the WT GFP protein.
But the most exciting was the finding of a blue FP (Y66H):
To sum up in his words:
“The availability of several forms of GFP with such different excitation and emission maxima [the most distinguishable pair being mutant P4 (Y66H) vs. mutant Pll (I167T)] should facilitate two-color assessment of differential gene expression, developmental fate, or protein trafficking. It may also be possible to use these GFP variants analogously to fluorescein and rhodamine to tag interacting proteins or subunits whose association could then be monitored dynamically in intact cells by fluorescence resonance energy transfer (19, 20). Such fluorescence labeling via gene fusion would be site-specific and would eliminate the present need to purify and label proteins in vitro and microinject them into cells.”
He saw the future, and it was bright green.
Heim, R., Prasher, D., & Tsien, R. (1994). Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proceedings of the National Academy of Sciences, 91 (26), 12501-12504 DOI: 10.1073/pnas.91.26.12501