Effects of counter ion protonation on the ultrafast excited-state dynamics in archaerhodopsin-3

Archaerhodopsin-3 (AR-3) and its mutants have attracted large attention for applications in optogenetics since they display a voltage-sensitive, near-infrared fluorescence. Recent quantum chemistry modeling of the electrostatic interactions between the protein environment and the protonated Schiff base retinal gave a detailed insight about the origin of a mutation-induced excited-state barrier, which hinders the usual subpicosecond photo-isomerization and increases the fluorescence quantum yield (Φ_f). However, despite the large range of mutations tested, the Φ_f rarely exceeds 1%, which is in large contrast to the sole naturally fluorescent retinal protein Neorhodopsin (Φ_f = 20%). In order to obtain a deeper insight into the electrostatic protonated Schiff base retina-protein interactions of wild-type (WT) AR-3, the present contribution sets out to identify the pK_a values of the neighboring aspartic acids D95 and D222, and hence their pH-dependent protonation states. This is done by a combined experimental and theoretical approach, which investigates the pH-dependent absorption spectra, isomer compositions, and excited-state lifetimes in WT AR-3, recombinantly expressed in E. coli. The mutant D95N with a neutral asparagine residue at position 95 is also studied as a reference system. We show that WT AR-3 displays two pK_a values at 1.5± 0.2 and 2.5± 0.2, which are related to the protonation of D222 and D95, respectively. One major argument for this assignment is the fact that the isomer composition and spectroscopic properties are the same for WT AR-3 at pH 2 and for the D95N mutant. Two different simulation approaches are used to describe the pH and mutation-induced effects, providing very good quantitative agreement in most cases. The benefits and limitations of each method are discussed in detail.