International Photochemistry Conference edition:2013 location:Leuven date:21-26 July 2013
One of the greatest tools to study the complicated cellular machinery at the micro- and nanometer scale is optical microscopy. The discovery and development of fluorescent proteins, and recent advances in diffraction-unlimited far-field optical microscopy have truly revolutionized our understanding of life and disease. While considerable resources are being spent on advanced equipment, the labels still remain a severely limiting factor. Especially for diffraction-unlimited microscopy, the demands are high.
We previously reported on NijiFP, a four-way highlighter FP that is not only green-to-red photoconvertible, but also reversibly photoswitching in both states. In the present work, we engineered a similar optical highlighter probe, this time by engineering green-to-red photoconversion properties into the well-known reversibly photochromic protein Dronpa.
We made ffDronpa, a Dronpa mutant that is formed up to three times as fast as Dronpa, while retaining the interesting photochromic features of Dronpa. Using rational and random mutagenesis, we transformed ffDronpa to pcDronpa. This Dronpa mutant combines Dronpa’s photochromism with the feature of being photoconvertible to a red state, emitting at 579nm. pcDronpa was studied in detail and was applied in several microscopic settings.
We have shown that atomic level structure determination plays a pivotal role in linking spectroscopic characteristics to structural features. Thus, single X-ray crystallography is highly integrated in our rational design of improved fluorescent probes. By inspecting the crystal structure, we were able to declare pcDronpa’s spectroscopic anomalies and have a greater understanding of structural features present in optical highlighter proteins. Moreover, this data led to structure-guided design of improved pcDronpa variants.
Our integrated, structure-guided design strategy for intelligent highlighter probes creates a paradigm for fluorescent protein design. Our example indeed demonstrates the elegant introduction of different photodynamic behaviors in a single protein scaffold, in our case Dronpa. This work will open up new possibilities for the design of FPs and tailoring their properties.