Functional importance of the oligomer formation of the cyanobacterial H+ pump Gloeobacter rhodopsin

Abstract

Many microbial rhodopsins self-oligomerize, but the functional consequences of oligomerization have not been well clarified. We examined the effects of oligomerization of a H + pump, Gloeobacter rhodopsin (GR), by using nanodisc containing trimeric and monomeric GR. The monomerization did not appear to affect the unphotolyzed GR. However, we found a significant impact on the photoreaction: The monomeric GR showed faint M intermediate formation and negligible H + transfer reactions. These changes reflected the elevated pKa of the Asp121 residue, whose deprotonation is a prerequisite for the functional photoreaction. Here, we focused on His87, which is a neighboring residue of Asp121 and conserved among eubacterial H + pumps but replaced by Met in an archaeal H + pump. We found that the H87M mutation removes the “monomerization effects”: Even in the monomeric state, H87M contained the deprotonated Asp121 and showed both M formation and distinct H + transfer reactions. Thus, for wild-type GR, monomerization probably strengthens the Asp121-His87 interaction and thereby elevates the pKa of Asp121 residue. This strong interaction might occur due to the loosened protein structure and/or the disruption of the interprotomer interaction of His87. Thus, the trimeric assembly of GR enables light-induced H + transfer reactions through adjusting the positions of key residues.

A unique clade of light-driven proton-pumping rhodopsins evolved in the cyanobacterial lineage

“…The M-decay rate constant at pH 7.0 was estimated to be 0.016 ms −1 by fitting a single exponential equation. This decay rate constant was much smaller than that reported for PR (4 ms −1 ) 25 , BR (0.25 ms −1 ) 35 , and GR (2.3 ms −1 and 1 ms −1 ) 15 , 36 indicating that the M-intermediate of N2098R was much longer-lived than that of these other rhodopsins (Table 2 ). When the same experiment was repeated under various pH conditions, the M-decay rate was markedly increased under acidic conditions, suggesting that proton concentration mediates the rate of decay of the M-intermediate (Supporting Information Fig.…”

“…3d and e). From the time and location of the absorption maxima, the increases and decreases at around 620, 405, and 645 nm were attributed to the formation and decay of the K-, M-, and O-intermediates, 15,36 indicating that the M-intermediate of N2098R was much longer-lived than that of these other rhodopsins (Table 2). When the same experiment was repeated under various pH conditions, the M-decay rate was markedly increased under acidic conditions, suggesting that proton concentration mediates the rate of decay of the M-intermediate (Supporting Information Fig.…”

Replaceability of Schiff base proton donors in light-driven proton pump rhodopsins

“…For expression and purification, E. coli strains BL21(DE3), UT5600, and BL21 were used for the wildtype and mutant DR, PR, and ESR, respectively. The respective procedures were essentially the same as those previously described ( 23 , 35 , 36 ). Briefly, the cells were grown in 2× YT medium at 37 °C, and expression was induced in the presence of 10 μM all- trans retinal by the addition of 1 mM isopropyl-β-D-thiogalactopyranoside for DR and ESR or 0.2% l -(+)-arabinose for PR.…”

Electronic Couplings and Electrostatic Interactions Behind the Light Absorption of Retinal Proteins

“…ECCD bands also appear in biomolecules, and a typical example is the negative and positive CD bands observed in the bacteriorhodopsin (BR) trimer ( Cassim, 1992 ; Pescitelli and Woody, 2012 ). However, there are few reports of ECCD in other biomolecules ( Tsukamoto et al, 2013 ; Pescitelli et al, 2014 ; Iizuka et al, 2019 ), and the details of the mechanism are not well understood.…”