Functional Green-Tuned Proteorhodopsin from Modern Stromatolites.

Virginia Helena Albarracín,Phillip G Wood,Ivana Kraiselburd,María Eugenia Farias,Wolfgang Gärtner,Christian Bamann,Ernst Bamberg,Erika Kothe

doi:10.1371/journal.pone.0154962

Abstract

The sequenced genome of the poly-extremophile Exiguobacterium sp. S17, isolated from modern stromatolites at Laguna Socompa (3,570 m), a High-Altitude Andean Lake (HAAL) in Argentinean Puna revealed a putative proteorhodopsin-encoding gene. The HAAL area is exposed to the highest UV irradiation on Earth, making the microbial community living in the stromatolites test cases for survival strategies under extreme conditions. The heterologous expressed protein E17R from Exiguobacterium (248 amino acids, 85% sequence identity to its ortholog ESR from E. sibiricum) was assembled with retinal displaying an absorbance maximum at 524 nm, which makes it a member of the green-absorbing PR-subfamily. Titration down to low pH values (eventually causing partial protein denaturation) indicated a pK value between two and three. Global fitting of data from laser flash-induced absorption changes gave evidence for an early red-shifted intermediate (its formation being below the experimental resolution) that decayed (τ1 = 3.5 μs) into another red-shifted intermediate. This species decayed in a two-step process (τ2 = 84 μs, τ3 = 11 ms), to which the initial state of E17-PR was reformed with a kinetics of 2 ms. Proton transport capability of the HAAL protein was determined by BLM measurements. Additional blue light irradiation reduced the proton current, clearly identifying a blue light absorbing, M-like intermediate. The apparent absence of this intermediate is explained by closely matching formation and decay kinetics.

Highlights

Microbial rhodopsins are considered to be the simplest energy-harvesting photoreceptors consisting of a single, membrane-embedded protein covalently binding a retinal (Vitamin-A aldehyde) chromophore [1]
E17R, a gene predicted as belonging to the bacteriorhodopsin family (L479_RS10205/ WP_016509804), was found in the genome of Exiguobacterium sp
E17R has 85% sequence identity with the green-PR of the psycrophilic Exiguobacterium sibiricum (ESR) for which the crystal structure was recently solved (PDB: 4HYJ) [21]. Alignment of these two proteins sequences with that of the blue-light absorbing proteorhodopsin (BPR) of uncultured Gamma-proteobacterium Hot 75m4 clearly showed the instrumental difference in an amino acid position associated with spectral tuning

Summary

Introduction

Microbial rhodopsins are considered to be the simplest energy-harvesting photoreceptors consisting of a single, membrane-embedded protein covalently binding a retinal (Vitamin-A aldehyde) chromophore [1]. The study of the microbial archetype, bacteriorhodopsin (BR) from the archaeon Halobacterium salinarum [2], led to the suggestion that BR evolved as a PLOS ONE | DOI:10.1371/journal.pone.0154962 May 17, 2016 proteorhodopsin from Modern Stromatolites collection and analysis, decision to publish, or preparation of the manuscript

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Conclusion