Voltage Mediated Control of Fluorescence Protein Emissions at the Metal-Solution Interface

Abstract

The fluorescence protein technologies have made remarkable contributions to the advancement of life science. Accordingly, the physicochemical properties of fluorescence proteins have been deeply investigated in the bulk solution that mimics the cellular environment, but those at the less common environment such as surface and interface have not been deeply investigated. We recently found the phenomenon that the fluorescence protein immobilized at the metal-solution interface exhibits voltage-dependent photoluminescence. Upon the blue light photoexcitation of Venus, a yellow-emitting variant of green fluorescence protein, immobilized on the gold electrode surface, robust enhancement or decrease of fluorescence was induced by applying negative or positive bias, respectively. This previously unappreciated phenomenon was then implemented as a protein-based microdisplay. We then sought to solve the mechanism for the cathodic enhancement utilizing the characteristic optical properties in the three different fluorescence proteins. From the simultaneous electrochemical and fluorescence measurements in Venus, we found a strong correlation between the fluorescence modulation and the current reflecting cathodic hydrogen evolution, which led to a hypothesis that shift in the protonation-deprotonation equilibrium of the chromophore driven by hydrogen evolution at the metal surface underlies the phenomena. The hypothesis predicted that voltage dependency should be also found in the green-to-red photoconversion of fluorescence protein which is known as a protonation-dependent process. The hypothesis was verified by observing clear voltage dependency for the photoconversion in KikGR, an engineered photoconvertible fluorescence protein, at the interface. We then addressed how the shift in protonation equilibrium is driven by hydrogen evolution. The analysis using iR-phuruolin, a fluorescence protein variant with the inverse pH-sensitivity revealed the existence of an interface-specific mode of protonation-deprotonation reactions, where the protonation equilibrium is directly coupled to the cathodic hydrogen evolution. The interface-specific mode is distinct from that conventionally seen in protein in the bulk solution where the protonation patterns of the constituent titratable residues are determined through the local environmental acid-base equilibrium. The potential applications based on this interface-specific mechanism are then discussed, including the spatially resolved monitoring of hydrogen evolution reactions at the near-neutral condition. Fluorescent Protein The original Green Fluorescent Protein (GFP) was discovered by the researchers back in the early 1960s when studying the bioluminescent properties of a blue light emitting bioluminescent protein called aequorin together with another protein isolated from Aequorea victoria jellyfish and that another protein was eventually named the Green Fluorescent Protein [1]. GFP like fluorescent proteins have been discovered in other organisms including corals, copepods, sea anemones, lancelets, zoanithids [2]. GFP can be modified because the genetic code and amino acid code is well known [3]. Modifications allow for GFP to fluoresce with different colors such as blue (BFP), yellow (YFP), cyan (CFP), red (RFP) [4]. Their physiochemical phenomena can be used for various biological and medical research [5,6]. Advances such as, Förster resonance energy transfer (FRET), a fluorescence microscopy application was developed with GFP and permitting the researchers to use even more specific and powerful applications of fluorescence for their imaging [7]. last ten years, many new RSFPs (Reversibly photoswitchable fluorescent proteins) have been developed and novel applications in cell imaging discovered that rely on their photoswitching properties [8,9].