Pro219 is an electrostatic color determinant in the light-driven sodium pump KR2

Abstract

Color tuning in animal and microbial rhodopsins has attracted the interest of many researchers, as the color of their common retinal chromophores is modulated by the amino acid residues forming the chromophore cavity. Critical cavity amino acid residues are often called “color switches”, as the rhodopsin color is effectively tuned through their substitution. Well-known color switches are the L/Q and A/TS switches located in the C and G helices of the microbial rhodopsin structure respectively. Recently, we reported on a third G/P switch located in the F helix of the light-driven sodium pumps of KR2 and JsNaR causing substantial spectral red-shifts in the latter with respect to the former. In order to investigate the molecular-level mechanism driving such switching function, here we present an exhaustive mutation, spectroscopic and computational investigation of the P219X mutant set of KR2. To do so, we study the changes in the absorption band of the 19 possible mutants and construct, semi-automatically, the corresponding hybrid quantum mechanics/molecular mechanics models. We found that the P219X feature a red-shifted light absorption with the only exception of P219R. The analysis of the corresponding models indicate that the G/P switch induces red-shifting variations via electrostatic interactions, while replacement-induced chromophore geometrical (steric) distortions play a minor role. However, the same analysis indicates that the P219R blue-shifted variant has a more complex origin involving both electrostatic and steric changes accompanied by protonation state and hydrogen bond networks modifications. These results make it difficult to extract simple rules or formulate theories for predicting how a switch operates without considering the atomistic details and environmental consequences of the side chain replacement.

Evolution of the Automatic Rhodopsin Modeling (ARM) Protocol

“…Considering that the wavelength dependence of the absorption efficiency defines the colors of the rhodopsin proteins, the modulation of the gap makes possible the engineering of either blue- or red-shifted rhodopsin variants, absorbing in specific regions of the UV-Vis spectrum (i.e., color tuning, see Sect. 6.4 ) [ 19 – 21 , 25 – 35 ]. Photoexcitation to the state can also be achieved by simultaneous absorption of two infrared photons (i.e., process) of the same energy E , corresponding to half of the energy necessary for [ 23 , 36 – 40 ].…”

“…(Most of the content of the following four sections is reproduced/adapted with permission from [ 69 ], copyright 2019 American Chemical Society, while content of Sect. 4.5 is reproduced/adapted with permission from [ 35 ], open access under a CC BY license (Creative Commons Attribution 4.0 International License)).…”

“…For instance, Fig. 10 illustrates how the customization, in terms of either selection of side-chain conformations and protonation states, is achieved for the case of the microbial rhodopsin [ 35 , 69 , 70 ]. As observed, in the 3X3C X-ray structure [ 70 ], the residue Asp-116, considered as the main counterion (MC) of the , exhibits two side-chain conformations, namely, AAsp and BAsp, labeled with occupancy numbers 0.65 and 0.35, respectively.…”

“…As shown in the right panel of Fig. 10 , such customization is performed through a more rational assignment of the protonation states of the two aspartic acid residues forming the counterion complex of the , namely Asp-116 and Asp-251 [ 35 ]. The default model predicts that both aspartic acids are negatively charged.…”

“…However, as further discussed in Refs. [ 35 , 43 , 69 ], the presence of these two negative charges would outbalance the single positive charge of the , generating the large blue-shifted effect mentioned above. Accordingly, in the customized model the secondary counterion (SC) Asp-251 is, instead, protonated (i.e., neutral) to counterbalance the charge in the vicinity of the .…”