Color Change of Proteorhodopsin by a Single Amino Acid Replacement at a Distant Cytoplasmic Loop

Yoshitsugu, Maiko; Shibata, Mikihiro; Ikeda, Daisuke; Furutani, Yuji; Kandori, Hideki

doi:10.1002/ange.200705989

Cited by 11 publications

(45 citation statements)

References 32 publications

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“…Kandori and co-workers (14) have shown that a single mutation A178R in the EF loop of GPR, which is far away from the retinal binding site, causes a surprisingly large redshift and changes the pK a of the primary proton acceptor D97 (Fig. 1).…”

Section: Discussionmentioning

confidence: 97%

“…For example, one single point mutation L105Q in GPR, which is close to the chromophore, shifts the absorption maximum toward blue, whereas the corresponding Q105L mutation in blue absorbing PR causes a green shift (6,13). The recent discovery of a 20 nm redshift of the GPR absorption spectrum upon a single mutation A178R in the EF loop came therefore as a great surprise as the mutation site is far away from the retinylidene (14). This effect is highly position specific and has not been observed in BR (15).…”

Section: Introductionmentioning

confidence: 99%

“…pH-dependent stationary absorption spectra of GPR (A) and GPR A178R (B). The single point mutation A178R causes a general redshift of up to 13 nm(14). The pK a of the primary proton acceptor D97 changes from 7.0 for GPR to 7.7 for GPR A178R as determined by analyzing the absorption maximum upon titration of the primary proton acceptor (C).…”

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confidence: 99%

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The EF Loop in Green Proteorhodopsin Affects Conformation and Photocycle dynamics

Mehler

Scholz

Ullrich

et al. 2013

Biophysical Journal

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The proteorhodopsin family consists of retinal proteins of marine bacterial origin with optical properties adjusted to their local environments. For green proteorhodopsin, a highly specific mutation in the EF loop, A178R, has been found to cause a surprisingly large redshift of 20 nm despite its distance from the chromophore. Here, we analyze structural and functional consequences of this EF loop mutation by time-resolved optical spectroscopy and solid-state NMR. We found that the primary photoreaction and the formation of the K-like photo intermediate is almost pH-independent and slower compared to the wild-type, whereas the decay of the K-intermediate is accelerated, suggesting structural changes within the counterion complex upon mutation. The photocycle is significantly elongated mainly due to an enlarged lifetime of late photo intermediates. Multidimensional MAS-NMR reveals mutation-induced chemical shift changes propagating from the EF loop to the chromophore binding pocket, whereas dynamic nuclear polarization-enhanced (13)C-double quantum MAS-NMR has been used to probe directly the retinylidene conformation. Our data show a modified interaction network between chromophore, Schiff base, and counterion complex explaining the altered optical and kinetic properties. In particular, the mutation-induced distorted structure in the EF loop weakens interactions, which help reorienting helix F during the reprotonation step explaining the slower photocycle. These data lead to the conclusion that the EF loop plays an important role in proton uptake from the cytoplasm but our data also reveal a clear interaction pathway between the EF loop and retinal binding pocket, which might be an evolutionary conserved communication pathway in retinal proteins.

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Section: Discussionmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 99%

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The EF Loop in Green Proteorhodopsin Affects Conformation and Photocycle dynamics

Mehler

Scholz

Ullrich

et al. 2013

Biophysical Journal

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“…It has been reported that these amino acid residues also affect protein folding functions and stability [27]. Ala178 in the distant cytoplasmic loop (E–F loop) affects the colour change of PR [28]. In addition, research on red‐shifted rhodopsin has been performed via a machine learning‐based experimental design method [29,30].…”

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confidence: 99%

Mutational analyses identify a single amino acid critical for colour tuning in proteorhodopsins

et al. 2022

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Microbial rhodopsins are light‐activated proteins that contain seven transmembrane alpha‐helices. Spectral tuning in microbial rhodopsins is a useful optogenetic tool. In this study, we report a new site that controls spectral tuning. In the proteorhodopsins ISR34 and ISR36, a single amino‐acid substitution at Cys189 caused an absorption maximum shift of 44 nm, indicating spectral tuning at a specific site. Comparison of single amino acid substitutions was conducted using photochemical and photobiological approaches. The maximum absorption for red‐shift was measured for mutations at positions 189 and 105 in ISR34, both residues being equally important. Structural changes resulting from amino acid substitutions are related to pKa values, pumping activity and spectral tuning.

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“…It is also possible that the amino acid replacement may have caused broader structural changes to the protein. In the case of proteorhodopsin, the replacement of A178, an amino acid located in the EF loop, with arginine caused large changes to absorption spectrum and photocycle of it . These changes include 20 nm redshift of the absorption maximum of the ground state, elevated pKa of the primary proton acceptor, a faster decay of K and M intermediates, 10 times elongation of the photocycle, and chromophore configurational change.…”

Section: Discussionmentioning

confidence: 99%

Two Consecutive Polar Amino Acids at the End of Helix E are Important for Fast Turnover of the Archaerhodopsin Photocycle

Geng

Dai

Chao

et al. 2019

Photochem & Photobiology

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Archaerhodopsins (ARs) is one of the members of microbial rhodopsins. Threonine 164 (T164) and serine 165 (S165) residues of the AR from Halorubrum sp. ejinoor (HeAR) are fully conserved in ARs, although they are far from the proton transfer channel and the retinal Schiff base, and are likely involved in a hydrogen-bonding network at the end of the Helix E where most microbial rhodopsins assume a "bent structure". In the present work, T164 and/or S165 were replaced with an alanine (A), and the photocycles of the mutants were analyzed with flash photolysis. The amino acid replacements caused profound changes to the photocycle of HeAR including prolonged photocycle, accelerated decay of M intermediate and appearance of additional two intermediates which were evident in T164A-and T164A/S165A-HeAR photocyles. These results suggest that although T164 and S165 are located at the far end of the photoactive center, these two amino acid residues are important for maintaining the fast turnover of the HeAR photocycle. The underlying molecular mechanisms are discussed in relation to hydrogenbonding networks involving these two amino acids. Present study may arouse our interests to explore the functional role of the well-conserved "bent structure" in different types of microbial rhodopsin.

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Color Change of Proteorhodopsin by a Single Amino Acid Replacement at a Distant Cytoplasmic Loop

Cited by 11 publications

References 32 publications

The EF Loop in Green Proteorhodopsin Affects Conformation and Photocycle dynamics

The EF Loop in Green Proteorhodopsin Affects Conformation and Photocycle dynamics

Mutational analyses identify a single amino acid critical for colour tuning in proteorhodopsins

Two Consecutive Polar Amino Acids at the End of Helix E are Important for Fast Turnover of the Archaerhodopsin Photocycle

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