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

Yuta Nakajima,Hideki Kandori,Massimo Olivucci,Laura Pedraza-González,Keiichi Inoue,Leonardo Barneschi

doi:10.1038/s42003-021-02684-z

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.

Highlights

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
We attempted to express the 19 different P219X mutants of KR2 in E. coli where all-trans retinal was added at the induction period to produce the rPSBAT chromophore
We have presented a combined experimental and computational investigation of the P219X mutants of KR2 supporting the existence of a G/P switch in microbial rhodopsins

Summary

Introduction

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. Here we refer to it as the “A/ TS switch”, as it appears to represent an additional example of naturally occurring color determining residue This switch satisfies the general principle of color tuning, where introduction of a polar residue in the vicinity of the β-ionone ring or the Schiff base moiety of the chromophore causes spectral red and blue shift, respectively[14,23,24,25]. This observation is in line with the present theoretical understanding of color tuning based on the electronic structure and, opposite positive charge distribution, of the ground (S0) and first excited (S1) states of rPSBAT. The exact mechanism that is at the basis of such color tuning effect is unknown

Methods

Results

Conclusion