Photobleaching and Stability of Red Fluorescent Proteins

Abstract

Fluorescent proteins (FPs) are used routinely to visualize structural organization and protein dynamics. Compared with other mutants, there is a large demand for red FPs (RFPs) due to the higher transparency of cells and tissue at longer wavelengths. However, the application of RFPs is limited by the increased susceptibility to photobleaching [1]. A possible mechanism for this may be a decrease in structural stability of the beta-barrel, leading to dark state conversion of the chromophore or oxygen access. To characterize the relation between photostability and structural stability, we use temperature-dependent terahertz (THz) time-domain spectroscopy [2]. Temperature dependent terahertz absorbance measurements were made between 80-270K as a function of photobleaching for mCherry, TagRFP-T and mOrange2 RFP samples. We find that: 1) the absolute THz response follows the thermal stability, as defined by the melting temperature; 2) the protein dynamical transition temperature [3] also follows the thermal stability; 3) the thermal stability increases with photobleaching and 4) the photostability does not follow the thermal stability. The THz sensitivity to thermal stability is verified by CD measurements. The higher stability with photobleaching is surprising, but could possibly be a driving force toward the photobleached state. Our result provides additional insight into photobleaching mechanism, and introduces a way to estimate the qualities of FPs.