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Biomedical Optics & Medical Imaging

Two-photon spectroscopy explains red fluorescent-protein color hues

Absorption wavelength changes are caused by variations in the internal electric field of the proteins.
19 January 2010, SPIE Newsroom. DOI: 10.1117/2.1200912.002541

The discovery of green fluorescent protein (GFP) has generated, within roughly a decade, a wide variety of fluorescent proteins (FPs) of almost any imaginable color hue. Genetically encoded fluorescent sensors are indispensable tools for imaging living cells and tissues. The large color variations, currently ranging from deep blue to far red, are caused by different FP-chromophore structures. Similarly to regular organic dye molecules, where changes in chemical structure tune the transition energy between the ground and excited states, new FP color hues are routinely generated through mutations that slightly alter the conjugation pathways within the FP chromophore.

However, crystallography shows that in some mutant FPs the chromophore structure remains the same although significant hue variations are observed. The most striking example of this unusual behavior occurs in the Fruit series of mutated red-FP variants (including mTangerine, mStrawberry, mCherry, tdTomato, mBanana, etc.). The relevant mutations are located exclusively in the staves of the surrounding beta-barrel protein, leaving the conjugation pathways of the red FP chromophore intact. Nevertheless, the absorption wavelengths show a large, ~50nm shift, from 540 to 590nm (corresponding shifts are also observed in fluorescence). How can perturbations occurring some 1nm away from the actual chromophore cause such dramatic changes in the transition wavelength?

Figure 1. Signature of the quadratic Stark effect in red fluorescent (FPs) proteins (labeled), showing the shift of the one-photon-absorption wavelength (vertical axes) as a function of measured dipole-moment difference of the lowest-energy S0-S1 transition (horizontal axes). All FPs on the parabolic curve share the same chromophore structure. pH: Measure of acidity or basicity of a solution. mRFP: Mutated red FP.

It has long been speculated that proteins and other biological macromolecules may generate strong electric fields because of their charged constituents. However, because local charges are tightly confined to specific nanoscale protein environments, detecting these fields has remained elusive. Can these internal fields be measured?

Surprisingly, both questions were recently resolved simultaneously in an experiment that proved that color hues in fruits are caused by the quadratic (second-order) Stark effect, the shifting and splitting of atomic and molecular spectral lines caused by these strong internal electric fields.1 Our team published results of three studies that formed the basis for this breakthrough. First, we studied how the intrinsic femtosecond two-photon-absorption (2PA) cross section in a variety of red FPs (including the Fruit series) depends on excitation wavelength.2 Contrary to previous assumptions, we observed that this 2PA wavelength dependence does not follow that of the corresponding one-photon absorption (1PA) and that 2PA spectra of the Fruits exhibit distinctive properties. Second, we developed improved experimental techniques, enabling measurements of the absolute 2PA cross sections.3 We used a femtosecond optical parametric amplifier (TOPAS-C, pumped by a coherent Legend-H femtosecond titanium:sapphire amplifier), which provided the required broad wavelength tunability (550–1800nm). Third, and perhaps decisively, we found that under certain conditions the 2PA cross section depends on the permanent electrical dipole moment (or, more accurately, on the difference of the permanent dipole moment in the ground and excited states) of the relevant transition.4 In particular, we showed that, by measuring the absolute 2PA cross section in the lowest-energy 0–0 transition, one can determine the value of the permanent electrical-dipole difference (provided that the corresponding transition-dipole moment is also known).

While studying the 2PA spectra of the Fruit series,2 we noticed that the peak transition frequency of the 1PA band correlates with the permanent electric-dipole-moment difference (see Figure 1).4 A quadratic dependence of the transition frequency on dipole moment is apparent, which implies that both must change systematically. Our data analysis led us to conclude that the different color hues of red FPs are most likely caused by an internal quadratic Stark effect. In addition, the second-order fit yields an effective internal electric-field value in each protein of between 10 and 100MV/cm.

It appears that the striking beauty of coral reefs, in both the variety of colors they contain and the way we perceive them, involves the effects of strong electric fields occurring within a nanoscopic protein environment: both the opsin proteins in the eye of the beholder and the FPs' beta barrels shape perception through the Stark effect. Our next steps will focus on rational FP design with increased two-photon brightness and improved photostability, and may eventually also provide improved insights into how living material functions and develops.

Aleks Rebane, Mikhail Drobizhev, Nikolay Makarov
Department of Physics
Montana State University
Bozeman, MT

Aleks Rebane obtained his PhD (1986) from the Institute of Physics in Tartu (Estonia), and his habilitation in physical chemistry from the Swiss Federal Institute of Technology, Zurich (1995). He was awarded the International Commission for Optics Prize in 1993 for his work on time-space holography. He has been a professor at Montana State University since 1997.

Mikhail Drobizhev obtained his MS (1986) from the Moscow Institute of Physics and Technology (Russia) and his PhD (1998) from the P. N. Lebedev Institute of Physics of the Russian Academy of Sciences in Moscow. He has been a research professor since 1999.

Nikolay Makarov received his MS (2003) from St. Petersburg State University of Information Technologies, Mechanics, and Optics (Russia). He is currently a PhD student. In 2008 he received a D. J. Lovell SPIE scholarship.

Thomas E. Hughes, Shane Tillo
Department of Cell Biology and Neuroscience
Montana State University
Bozeman, MT

Thomas Hughes received his BS in biology (1981) from Tufts University and a PhD in neuroanatomy (1986) from Duke University. He is currently a professor and head of the Department of Cell Biology and Neuroscience.

Shane Tillo received his BS in cell biology and neuroscience from Montana State University in 2007. He is currently receiving graduate training at the Vollum Institute for Neuroscience at the Oregon Health and Science University.