Professor, Department of Anatomy & Structural Biology
Non-invasive optical imaging, monitoring and manipulation of metabolic processes in living mammals is more feasible within the near-infrared (NIR) optical transparency window (650-900 nm) where hemoglobin and melanin absorbance significantly decreases, and water absorbance is still low. The most red-shifted fluorescent proteins (FPs) of the GFP-like family have excitation and emission spectra outside of the NIR region and suffer from low brightness and modest photostability. Natural bacterial phytochrome photoreceptors (BphPs) utilize an enzymatic product of heme, low-molecular weight biliverdin, as a chromophore.
BphPs provide many advantages over other natural chromophore containing proteins. Unlike the chromophores of non-bacterial phytochromes, biliverdin is ubiquitous in mammals. This makes BphP applications in mammalian cells, tissues and whole mammals as easy as conventional GFP-like FPs, without supplying chromophore through an external solution. BphPs exhibit NIR absorbance and fluorescence, which are red-shifted relative to that of any other phytochromes, and lie within the NIR optical window. This makes BphPs spectrally complementary to other existing optical probes and optogenetic tools based on the GFP, flavoprotein and rhodopsin-like protein families. Independent domain architecture and pronounced conformational changes upon biliverdin photoisomerization make BphPs attractive templates to design various photocontrollable genetically-encoded probes.
In our laboratory, we engineer new BphP-based FPs, biosensors and optogenetic tools. These include bright and spectrally resolvable permanently fluorescent NIR FPs, photoactivatable with non-phototoxic NIR light FPs, and reversibly photoswitchable FPs. We also focus on designing of NIR reporters for protein interactions and biosensors for intracellular ions and metabolites. Lastly, we engineer BphPs into optogenetic elements allowing to noninvasively regulate intracellular processes in vivo with NIR light.
We apply various directed protein evolution approaches based on rational structure-based design and random mutagenesis of template BphPs, high-throughput flow cytometry and multiwell plate spectroscopy. These conventional techniques allow screening for standard properties of genetically-encoded probes, such as excitation and emission wavelengths, brightness, photostability, pH stability and folding efficiency. We also develop new protein engineering and high-throughput approaches to specifically optimize BphP-based constructs. These include time-resolved fluorescence lifetime measurements, expression in bacterial periplasmic space, screening of mutant libraries in yeast and in mammalian cells using shuttle vectors and inducible somatic hypermutations.
The resulting NIR probes, biosensors and molecular tools are tested in mouse models and applied to various in vivo studies. These NIR constructs extend optical methods to multicolor deep-tissue in vivo imaging, cell and tissue labeling, photoactivation and tracking, and detection of enzymatic activities and protein interactions in cells, tissues and whole mammals. The engineered NIR optogenetic tools allow light-manipulations of cellular processes directly through the skin of living animals.
1. Shcherbakova D.M., and Verkhusha V.V. Near-infrared fluorescent proteins for multicolor in vivo imaging. Nature Methods 2013, 10: 751-754.
2. Piatkevich, K.D., Subach F.V. and Verkhusha V.V. Far-red light photoactivatable near-infrared fluorescent proteins engineered from a bacterial phytochrome. Nature Communications 2013, 4: 2153.
3. Piatkevich K.D., Subach F.V. and Verkhusha V.V. Engineering of bacterial phytochromes for near-infrared imaging, sensing, and light-control in mammals. Chemial Society Reviews 2013, 42: 3441-3452.
4. Subach F.M. and Verkhusha V.V. Chromophore transformations in red fluorescent proteins. Chemical Reviews 2012, 112: 4308-4327.
5. Subach F.V., Piatkevich K.D., and Verkhusha V.V. Directed molecular evolution to design advanced red fluorescent proteins. Nature Methods 2011, 8: 1019-1026.
6. Filonov G.S., Piatkevich K.D., Ting L.-M., Zhang J., Kim K., and Verkhusha V.V. Bright and stable near infra-red fluorescent protein for in vivo imaging. Nature Biotechnology 2011, 29: 757-761.
7. Subach O.M., Patterson G.H., Ting L.-M., Wang Y., Condeelis J.S., and Verkhusha V.V. A photoswitchable orange-to-far-red fluorescent protein, PSmOrange. Nature Methods 2011, 8: 771-777.
8. Piatkevich K.D., Hulit J., Subach O.M., Wu B., Abdulla A., Segall J.E., and Verkhusha V.V. Monomeric red fluorescent proteins with a large Stokes shift. Proc. Natl. Acad. Sci. USA 2010, 107: 5369-5374.
9. Subach F.V., Subach O.M., Gundorov I.S., Morozova K.S., Piatkevich K.D., Cuervo A.M., and Verkhusha V.V. Monomeric fluorescent timers that change color from blue to red report on cellular trafficking. Nature Chemical Biology 2009, 5: 118-126.
10. Subach F.V., Patterson G.H., Manley S., Gillette J.M., Lippincott-Schwartz J., and Verkhusha V.V. Photoactivatable mCherry for high-resolution two-color fluorescence microscopy. Nature Methods 2009, 6: 153-159.
More Information About Dr. Vladislav Verkhusha
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Albert Einstein College of Medicine
Jack and Pearl Resnick Campus
1300 Morris Park Avenue
Ullmann Building, Room 1217
Bronx, NY 10461
Nature Methods interviews Vladislav Verkhusha, Ph.D., regarding his work developing near-infrared (NIR) fluorescent proteins (FP) and their best research applications. (Free registration required.)