Proteorhodopsin Function Is Primarily Mediated by Oligomerization in Different Micellar Surfactant Solutions

Abstract

The diverse functionalities of membrane proteins (MPs) have garnered much interest in leveraging these biomolecules for technological applications. One challenge of studying MPs in artificial micellar surfactant environments is that many factors modulate their structures and functionalities, including the surfactants that interact with the MP or their assembly into oligomers. As oligomerization offers a means by which MPs could selectively interact among the copious environmental factors in biological environments, we hypothesized that MP function is predominantly modified by oligomerization rather than interactions with local surfactants that, by comparison, largely interact with MPs nonspecifically. To test this, we study the light-activated proton pump proteorhodopsin (PR) in micellar surfactant solutions because it is functionally active in monomeric and oligomeric forms, the light-activated functionalities of which can be assessed in detail. The surfactant composition and oligomerization are correlated with PR function, as measured by the protonation behaviors of aspartic acid residue 97, which mediates light-activated proton transport, and the associated photocycle kinetics. The results demonstrate that oligomerization dominantly mediates PR function in different surfactant environments, whereas some surfactants can subtly modulate proton-pumping kinetics. This work underscores the importance of understanding and controlling oligomerization of MPs to study and exploit their function.

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

“…Here, we found that the His-Asp interaction of GR becomes stronger upon monomerization and significantly increases the pKa of the Asp121 residue, which is fatal for the H + pump function. As mentioned above, a relatively small but similar pKa increase was also observed for PR upon monomerization 20,21 . Thus, the stronger His-Asp interaction might occur for both XR and PR.…”

“…Thus, higher pKa narrows the pH range where the H + pump exerts the activity. The observed pKa increase was approximately 1.1 unit 21 . The resultant pKa of 7.5–7.7 is still equal to or lower than the seawater pH of 7.6–8.2 22 .…”

“…Thus, the oligomeric state of BR is advantageous for higher activity but is not essential for the function itself 19 . Monomerization effects were also examined for the eubacterial H + pump proteorhodopsin (PR), and both advantages and disadvantages were reported 20,21 . The monomeric PR shows a faster photocycle, which means a faster turnover rate and thus elevates the total amount of transported H + .…”

Electrostatic Environment of Proteorhodopsin Affects the pKa of Its Buried Primary Proton Acceptor

“…PR pH-dependent optical absorbance change was recorded by a UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan), and the data analysis for getting the pKa D97 was done by the processing and fitting algorithm described in previous studies (15). Prepared PR-containing liposomes were diluted by the HEPES buffer with the desired salt concentrations to achieve a total volume of 750 mL and an initial optical density between 0.3 and 0.5 at 520 nm.…”

“…Moreover, the pKa of embedded residues was also shown to be affected by the environment in which the TMP resides. For example, the pKa of the embedded D97 residue (pKa D97 ) of PR can differ by more than one unit by the extent of oligomerization of surfactant-reconstituted PR (14,15), as well as the NaCl concentration in the buffer that contains detergent-constituted PR (16). These results unveiled that the external environment in which TMP resides can affect the pKa of embedded charged residues, but not the underlying mechanism.…”