pH-dependent transient conformational states control optical properties in cyan fluorescent protein

Abstract

A recently engineered mutant of cyan fluorescent protein (WasCFP) that exhibits pH-dependent absorption suggests that its tryptophan-based chromophore switches between neutral (protonated) and charged (deprotonated) states depending on external pH. At pH 8.1, the latter gives rise to green fluorescence as opposed to the cyan color of emission that is characteristic for the neutral form at low pH. Given the high energy cost of deprotonating the tryptophan at the indole nitrogen, this behavior is puzzling, even if the stabilizing effect of the V61K mutation in proximity to the protonation/deprotonation site is considered. Because of its potential to open new avenues for the development of optical sensors and photoconvertible fluorescent proteins, a mechanistic understanding of how the charged state in WasCFP can possibly be stabilized is thus important. Attributed to the dynamic nature of proteins, such understanding often requires knowledge of the various conformations adopted, including transiently populated conformational states. Transient conformational states triggered by pH are of emerging interest and have been shown to be important whenever ionizable groups interact with hydrophobic environments. Using a combination of the weighted-ensemble sampling method and explicit-solvent constant pH molecular dynamics (CPHMD(MSλD)) simulations, we have identified a solvated transient state, characterized by a partially open β-barrel where the chromophore pKa of 6.8 is shifted by over 20 units from that of the closed form (6.8 and 31.7, respectively). This state contributes a small population at low pH (12% at pH 6.1) but becomes dominant at mildly basic conditions, contributing as much as 53% at pH 8.1. This pH-dependent population shift between neutral (at pH 6.1) and charged (at pH 8.1) forms is thus responsible for the observed absorption behavior of WasCFP. Our findings demonstrate the conditions necessary to stabilize the charged state of the WasCFP chromophore (namely, local solvation at the deprotonation site and a partial flexibility of the protein β-barrel structure) and provide the first evidence that transient conformational states can control optical properties of fluorescent proteins.

Figure 1

A: Structure of WasCFP showing C_α positions of mutated residues (V61K, D148G, Y151N, L207Q). B: Structure of Trp66-based WasCFP chromophore covalently bound to β-barrel at positions showed with dashed lines (Leu64 and Val68). C: Deprotonation of CRF. CRF-H and CRF⁻ are neutral (protonated) and charged (deprotonated) forms of the synthetic chromophore, respectively.

Figure 2

Thermodynamic cycle that shows alchemical transformations considered in this study. Cyan and yellow-colored side chains correspond to V61 and K61 in WTP and V61KP peptides, respectively. pK_a values computed from CPHMD^MSλD simulations are highly elevated in both WT and WAS and, thus, are not responsible the observed pH-dependent absorption of WasCFP.

Figure 3

A: Probability distribution of the hydration parameter of RES in the WAS protein shows transiently populated states with large hydration parameters. B: Snapshots of the chromophore environment in four different conformations of WAS corresponding to hydration parameters φ=2, 7, 12, and 15. Number of water molecules within 7Å of nitrogen of the chromophore (3, 8, 15, and 17, respectively) and the corresponding pKa values computed using CPHMD^MSλD simulations are shown for each conformation.

Figure 4

Distances between C_α-C_α atoms of residues in β7 and β10 strands in the open state (dominant at pH=8.1) vs. the closed state (dominant at pH=6.1).

Figure 5

pH-dependent F^open computed using two (A) and three (C) state model. Correlation with F^open estimated from the pH-dependent absorption data is in (B) and (D), respectively.

Figure 6

Schematic illustration of the three-state model. K_prot and K_deprot are equilibrium constants corresponding to the pH-independent conformational transitions between open and closed states.