Color-changing mutation in the E-F loop of proteorhodopsin

Area of Science:

• Biochemistry
• Spectroscopy
• Protein Engineering

Background:

• Rhodopsins' color tuning is typically attributed to amino acids near the retinal chromophore.
• Proteorhodopsin (PR), found in marine bacteria, exhibits color shifts upon mutation at distant sites.
• The Ala178 residue in PR's E-F loop, ~25 Å from the chromophore, significantly impacts its absorption spectrum.

Purpose of the Study:

• To elucidate the molecular mechanism behind the color tuning effect of the Ala178Arg mutation in proteorhodopsin.
• To investigate the role of the E-F loop and specific positions within it on proteorhodopsin's spectral properties.
• To determine if this effect is specific to proteorhodopsin compared to bacteriorhodopsin.

Main Methods:

• Site-directed mutagenesis to introduce Arg at position 178 (A178R) and other positions in PR, and M163R in bacteriorhodopsin (BR).
• UV-Vis absorption spectroscopy to measure lambda(max) shifts.
• Fourier-transform infrared (FTIR) spectroscopy at 77 K to analyze chromophore structure and proton pumping activity.

Main Results:

• The A178R mutation in PR shifted lambda(max) from 525 nm to 545 nm, while M163R in BR showed no spectral change.
• Mutations at other positions in the PR E-F loop (T177R, N176R, S179R, P180R) had smaller or no spectral effects, indicating position-specificity.
• FTIR and proton pumping assays revealed similar chromophore structures and normal proton pumping for wild-type and A178R PR.

Conclusions:

• The E-F loop in proteorhodopsin possesses a unique structure that influences the transmembrane region.
• Mutation of Ala178 disrupts this unique E-F loop structure, leading to significant shifts in absorption spectra and pK(a).
• This study demonstrates that amino acids distant from the chromophore can play a crucial role in rhodopsin color tuning.