This project arose through the serendipitous meeting, and eventual collaboration, of three groups. The Rebane laboratory in physics has a long standing research program on two-photon absorption (2PA) properties of fluorescent dyes, the Hughes laboratory in cell biology has been working on fluorescent proteins and channels for two decades, and the Callis laboratory in chemistry has a deep theoretical understanding of 2PA and electrostatic properties of proteins. Initially the group came together to understand the mechanisms that shape 2PA in the fluorescent proteins. This led to the discovery that very strong electrostatic forces are shaping the absorption properties of the chromophore. This fascinating discovery was the direct result of an intermingling of biologists, chemists, and physicists at an intersection between their disciplines that was ripe for exploration. Here we are taking next logical step: determine if and how can we use the sensitivity of 2PA to measure electrostatics in complex proteins. If successful, the proposal may lead to a new kind of optical voltmeter that can measure forces in entirely new ways with unprecedented accuracy and fidelity. The unique requisites of our approach are: 7 The fields in proteins are measured under ambient conditions, indeed in living systems;7 Not rely on external fields or any kind of electrical contacts;7 Use all-optical detection compatible with existing optical microscopes;7 Operate with near-infrared light to minimize damage and provide deeper sample penetration;Under certain conditions the 2PA cross section is a function of the difference between electrical dipole moment in ground- and excited state, which in turn is a function of electric field strength and direction acting at the precise location. We take advantage of quantitative relation between the value of 2PA cross section and the electric field acting at the location of the chromophore in situ. This allows, for the first time, determining the electric filed inside proteins, membranes etc. from basic physical principles. A further appealing aspect of this method is that it meshes well with two-photon microscopes already strategically positioned in many biology research labs. The first specific aim is to develop a set of reference fluorescent dyes molecules, where we know exactly the molecular dipole moment dependence on the field from experimental measurements and quantum-chemical calculations. In the second aim, we will test our technique in simple model systems such as dyes incorporated into commercially-available proteins and artificial lipid bi-layers. Finally, we will covalently attach our probes to specific cysteins within the shaker potassium channel and measure the field within the channel in different conformations. We are attacking these challenges with an interdisciplinary team comprising internationally renowned experts in optical- and molecular physics, molecular biology and computational chemistry. If successful, this project will open up new frontiers in not only in molecular biology and study of biological macromolecules. Due to its inherent simplicity, we anticipate that our all- optical molecular voltmeter will be relatively straightforward to use, and may be applied to a broad range of problems.
We are proposing a new way of studying electrostatic properties of protein molecules using laser light. The goal is to observe how such molecules perform key functions in living cells on a nanometer scale. This new technique may enable diagnosis and treatment of diseases caused by malfunction of certain molecular mechanisms.
| Stoltzfus, Caleb R; Rebane, Aleksander (2016) High contrast two-photon imaging of fingermarks. Sci Rep 6:24142 |
| de Reguardati, Sophie; Pahapill, Juri; Mikhailov, Alexander et al. (2016) High-accuracy reference standards for two-photon absorption in the 680-1050 nm wavelength range. Opt Express 24:9053-66 |
| Stoltzfus, Caleb R; Rebane, Aleksander (2016) Optimizing ultrafast illumination for multiphoton-excited fluorescence imaging. Biomed Opt Express 7:1768-82 |
| Sarkisyan, Karen S; Goryashchenko, Alexander S; Lidsky, Peter V et al. (2015) Green fluorescent protein with anionic tryptophan-based chromophore and long fluorescence lifetime. Biophys J 109:380-9 |
| Friese, Daniel H; Mikhaylov, Alexander; Krzeszewski, Maciej et al. (2015) Pyrrolo[3,2-b]pyrroles-From Unprecedented Solvatofluorochromism to Two-Photon Absorption. Chemistry 21:18364-74 |
| Drobizhev, M; Callis, P R; Nifosì, R et al. (2015) Long- and Short-Range Electrostatic Fields in GFP Mutants: Implications for Spectral Tuning. Sci Rep 5:13223 |
| Guan, Yinghua; Meurer, Matthias; Raghavan, Sarada et al. (2015) Live-cell multiphoton fluorescence correlation spectroscopy with an improved large Stokes shift fluorescent protein. Mol Biol Cell 26:2054-66 |
| Stoltzfus, Caleb R; Barnett, Lauren M; Drobizhev, Mikhail et al. (2015) Two-photon directed evolution of green fluorescent proteins. Sci Rep 5:11968 |
| Rebane, Aleksander; Wicks, Geoffrey; Drobizhev, Mikhail et al. (2015) Two-photon voltmeter for measuring a molecular electric field. Angew Chem Int Ed Engl 54:7582-6 |
| Rebane, Aleksander; Drobizhev, Mikhail; Makarov, Nikolay S et al. (2014) Symmetry Breaking in Platinum Acetylide Chromophores Studied by Femtosecond Two-Photon Absorption Spectroscopy. J Phys Chem A 118:3749-3759 |
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