L105K mutant of proteorhodopsin

Area of Science:

• Microbial rhodopsins
• Biophysics
• Photochemistry

Background:

• Proteorhodopsin (PR) is a light-driven proton pump in marine bacteria.
• PR variants absorb blue light (λmax ~490 nm) or green light (λmax ~525 nm).
• Color is determined by amino acid at position 105 (Gln for blue, Leu for green), near the retinal chromophore.

Purpose of the Study:

• To investigate the effect of introducing a positive charge at position 105 in PR.
• To characterize the spectral, stability, and proton transport properties of the L105K mutant.

Main Methods:

• Site-directed mutagenesis to create the L105K PR mutant.
• Spectroscopic analysis (absorption, flash photolysis) to determine spectral shifts and intermediate decay kinetics.
• Thermal stability assays and hydroxylamine reaction tests.
• Measurement of light-induced proton pumping rates.

Main Results:

• The L105K mutant exhibited a ~21 nm red shift in absorption (λmax ~549 nm) at pH 7.0.
• The mutation caused slight structural destabilization but increased stability against hydroxylamine.
• Flash photolysis showed a ~3-fold slower M intermediate decay at pH 9.0.
• Light-induced proton transport rate was ~6 times slower in the L105K mutant compared to wild-type PR.

Conclusions:

• Introducing a positive charge (Lysine) at position 105 perturbs the proton transfer pathway, affecting the retinal Schiff base and proton donor.
• The L105K mutation significantly slows proton transport and alters the photocycle kinetics.
• This study provides the first report of a positive charge introduced into the hydrophobic cytoplasmic domain of microbial rhodopsins, impacting function.