Proton Wire Dynamics in the Green Fluorescent Protein

Abstract

Inside proteins, protons move on proton wires (PWs). Starting from the highest resolution X-ray structure available, we conduct a 306 ns molecular dynamics simulation of the (A-state) wild-type (wt) green fluorescent protein (GFP) to study how its PWs change with time. We find that the PW from the chromophore via Ser205 to Glu222, observed in all X-ray structures, undergoes rapid water molecule insertion between Ser205 and Glu222. Sometimes, an alternate Ser205-bypassing PW exists. Side chain rotations of Thr203 and Ser205 play an important role in shaping the PW network in the chromophore region. Thr203, with its bulkier side chain, exhibits slower transitions between its three rotameric states. Ser205 experiences more frequent rotations, slowing down when the Thr203 methyl group is close by. The combined states of both residues affect the PW probabilities. A random walk search for PWs from the chromophore reveals several exit points to the bulk, one being a direct water wire (WW) from the chromophore to the bulk. A longer WW connects the "bottom" of the GFP barrel with a "water pool" (WP1) situated below Glu222. These two WWs were not observed in X-ray structures of wt-GFP, but their analogues have been reported in related fluorescent proteins. Surprisingly, the high-resolution X-ray structure utilized herein shows that Glu222 is protonated at low temperatures. At higher temperatures, we suggest ion pairing between anionic Glu222 and a proton hosted in WP1. Upon photoexcitation, these two recombine, while a second proton dissociates from the chromophore and either exits the protein using the short WW or migrates along the GFP-barrel axis on the long WW. This mechanism reconciles the conflicting experimental and theoretical data on proton motion within GFP.

Ultrafast Oxidation of a Tyrosine by Proton-Coupled Electron Transfer Promotes Light Activation of an Animal-like Cryptochrome

“…•O hydrogen bond length of 2.7 Å ) in green fluorescent protein 91,92 but depends very strongly on the transfer distance, decreasing typically by one order of magnitude per 0.07 Å. 93 It is hence questionable that proton transfer from Y 373 H •+ to D 321can be faster than back ET from W 322 to Y 373 H •+ .…”

Mechanisms of ATP to cAMP Conversion Catalyzed by the Mammalian Adenylyl Cyclase: A Role of Magnesium Coordination Shells and Proton Wires

“…The importance of proton wires in protein systems is documented in previous works. [27][28][29][30][31][32][33][34] Simulations described here extend the experience with proton wires in the two-metal catalysis.…”

Computational Modeling Reveals the Mechanism of Fluorescent State Recovery in the Reversibly Photoswitchable Protein Dreiklang

“…Proton wires are important for understanding various properties of proteins including GFP. 22,[27][28][29] In this work, we do not discuss photochemical reactions in Dreiklang beyond a tentative mechanistic suggestion for the light-induced recovery reaction (Fig. 6).…”

“…It guarantees an efficient proton transfer along this proton wire. [27][28][29] In GFP, a similar proton wire connects the phenolic oxygen of Chro with the Oε(Glu222) via Ser205 and a water molecule. 22 Proton transfer along such a route is an essential part of the GFP machinery.…”