Pressure-Induced Changes in the Fluorescence Behavior of Red Fluorescent Proteins

Abstract

We present an experimental study on the fluorescence behavior of the red fluorescent proteins TagRFP-S, TagRFP-T, mCherry, mOrange2, mStrawberry, and mKO as a function of pressure up to several GPa. TagRFP-S, TagRFP-T, mOrange2, and mStrawberry show an initial increase in fluorescence intensity upon application of pressure above ambient conditions. At higher pressures, the fluorescence intensity decreases dramatically for all proteins under study, probably due to denaturing of the proteins. Small blue shifts in the fluorescence spectra with increasing pressure were seen in all proteins under study, hinting at increased rigidity of the chromophore environment. In addition, mOrange2 and mStrawberry exhibit strong and abrupt changes in their fluorescence spectra at certain pressures. These changes are likely due to structural modifications of the hydrogen bonding environment of the chromophore. The strong differences in behavior between proteins with identical or very similar chromophores highlight how the chromophore environment contributes to pressure-induced behavior of the fluorescence performance.

Exploring Structural and Optical Properties of Fluorescent Proteins by Squeezing: Modeling High-Pressure Effects on the mStrawberry and mCherry Red Fluorescent Proteins

“…Conf I yields consistent red-shift (−0.02 eV), whereas excitation energy of Conf II is blue-shifted by 0.13 eV. As described in the companion paper, 28 a small blue shift (0.005–0.01 eV) is observed with moderate pressure increase for all FPs from the mFruit family. At higher pressures, the spectra become broader, and some FPs exhibit larger shifts, i.e., mStrawberry exhibits large blue shift (+0.10 eV), whereas mOrange — a red one (−0.08 eV).…”

“…28 The pressure at which fluorescence reaches half its maximum value is 670 MPa and 300 MPa for mStrawberry and mCherry, respectively. 28 By understanding the differences between the two, we intend to identify structural elements that are essential for improved quantum yield. Modeling pressure-induced spectral shifts will help us to quantify the effect of neighboring residues, the structure of the chromophore and its binding pocket on the spectral properties of RFPs.…”

“…28 Towards this goal, we focus on the two representative FPs, mStrawberry and mCherry. While they share an identical chromophore, mStrawberry features an initial increase of fluorescence intensity reaching maximum (1.8 relative to ambient pressure) at 350 MPa, whereas fluorescence of mCherry only decreases.…”

Pressure Induced Structural Changes and Gas Diffusion Pathways in Monomeric Fluorescent Proteins

“…The difference response to pressure of these proteins are shown in Figure 4.1 (70). The fluorescence intensity of mStrawberry increases with the application of moderate pressure and decreases at higher pressure.…”

“…Recent high pressure experimental studies on the red fluorescent proteins mCherry and mStrawberry show that these proteins respond very differently to hydrostatic pressure (70), despite structural similarities in their protein barrels and in their chromophores. The difference response to pressure of these proteins are shown in Figure 4.1 (70).…”