“I then most always saw, with great wonder, that in the said matter there were many very little moving animalcules, very prettily a-moving.” -Antonie van Leeuwenhoek
No process has been more fundamental to the progress of biology than the careful observation of natural phenomena. It should, therefore, come as no surprise that among the most revolutionary historical discoveries in the field – inheritance and evolution, antibiotics, genetic engineering – stand new methods to observe the previously unseen world. Consider Robert Hooke and Antonie van Leeuwenhoek, who in the 1660’s developed new microscopes that allowed them to first observe cells. Or Osamu Shimomura, Martin Chalfie, and Roger Tsien, who in the late 20th century developed fluorescent proteins as a tool to visualize structures within living cells. Since the latter trio’s pioneering discoveries, the field of fluorescence microscopy has flourished, evolving to see smaller, faster, more diverse, and more dynamic events within the cell. Carrying on that tradition, lead author Dr. Jason Klima, a former graduate student in the lab of Dr. David Baker at the University of Washington’s Institute for Protein Design, in collaboration with research technician Lindsey Doyle and Dr. Barry Stoddard, professor in the Basic Sciences Division at Fred Hutch, in a recent article published in Nature Communications, engineered a suite of new molecular tools to shed light on various states within cells.
Classical fluorescent proteins, which fluoresce when exposed to specific wavelengths of light, are used to light up cells, or certain structures within cells, for observation. They have also been engineered to reflect changes within cells – to alter their fluorescence when, for instance, certain environmental conditions change, when specific genes turn on or off, or when biomolecular complexes come close together. But these proteins have their weaknesses. They are rather big and bulky, causing their mere presence to sometimes disturb the functions of the structures they are meant to reveal. And they can become unstable under certain conditions, or after illumination, and lose their ability to fluoresce. To overcome these limitations, the authors examined another type of fluorescent protein –mini-fluorescence-activating proteins (mFAPs), which do not themselves fluoresce but instead can bind to and activate the fluorescence of a compound called DFHBI. “mFAPs have several biophysical properties that make them attractive candidates for further development,” the authors noted. They are, for example, smaller and less perturbative of the structures to which they are bound, as well as more stable. The problem was that the promise of mFAPs, which have been only very recently developed, was still largely theoretical. The group therefore sought to engineer several new mFAPs to expand their functionality and utility.
“We began by seeking to improve the stability of mFAPs at low pH, the binding affinity to [DFHBI], as well as the fluorescence intensity,” said the authors. By testing libraries of mutant mFAPs, they found variants that were up to 17.5-fold brighter and up to 6.2-fold more photostable than an engineered green fluorescent protein. They then used this technique to develop two mFAPs that could report on important environmental conditions that change dynamically within cells – pH and calcium levels. The first variant they engineered, “mFAP_pH”, changed the excitation wavelength of its fluorescence based on how acidic or basic the environment was, allowing for a “real-time in situ quantification of pH” across a wide pH range. For the calcium reporters, the group engineered calcium-binding domains into the mFAP and identified variants whose fluorescence changed in response to calcium binding. Both the pH- and calcium-sensing mFAPs showed significant improvements in sensitivity and dynamic range over existing fluorescent protein reporters of these conditions. For a final challenge, the authors developed a “split mFAP” – a version of the protein that has been broken into two parts. Split proteins can only function when brought into contact and made whole again and are thus valuable reporters of when two molecules, each of which has been fused to one part of the split protein, come near one another. Again, using their protein design strategy, the group developed a highly functional pair of split mFAP fragments that could reveal when the proteins to which they were fused to came together and, importantly, when they separated, the latter being a limitation of some current protein technologies.
In designing mFAPs with several novel functions, the authors illuminated the promise of this new form of fluorescence-activating molecules to expand what events we can observe within living cells. “The engineering of useful fluorogenic sensors based on mFAPs is still in its early days,” they note. Adds Dr. Klima, "Moving forward, there are numerous exciting opportunities to engineer mFAPs to respond to proteins, metabolites and ions of interest for studying biology and creating new sensing devices. De novo designed mFAPs could [also] become useful tools for super-resolution fluorescence microscopy applications," another exciting and rapidly emerging field. Considering the pace of progress so far, these innovations are likely right around the corner.
This work was supported by the National Science Foundation, the National Institutes of Health, and the Howard Hughes Medical Institute.
Fred Hutch/UW Cancer Consortium members Barry Stoddard and David Baker contributed to this work.
Klima, J.C., Doyle, L.A., Lee, J.D. et al. (2021) Incorporation of sensing modalities into de novo designed fluorescence-activating proteins. Nat Commun 12, 856. https://doi.org/10.1038/s41467-020-18911-w