טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentAharonovich Dikla
SubjectBlue vs. Green Proteorhodopsin - Localizing the Factors
Responsible for the Difference in
Absorption Spectrum between Various
Proteorhodopsins
DepartmentDepartment of Biology
Supervisor Professor Oded Beja


Abstract

Proteorhodopsins (PR), ubiquitous retinylidene photoactive proton pumps, were recently discovered in the cosmopolitan uncultured SAR86 bacterial group in oceanic surface waters.  The PR gene encodes a polypeptide of 247 amino acids, with a molecular weight of 27 kD.  Similar to other rhodopsins, PR is formed from seven transmembrane α-helices.  The chromophore in the protein is all-trans retinal.  Photoisomeration to 13-cis initiates a photocycle, and a proton is transported across the cell membrane. 

Two related PR families, identified in the Pacific and the southern Oceans,

absorb light with different absorption maxima, 525 nm (green) and 490 nm (blue), and their distribution was shown to be stratified with depth, Green-PR with ‘green-absorbing’ pigments at the surface and Blue-PR with ‘blue-absorbing’ pigments at deeper waters in accordance with light available at these depths. The PR family is an ideal model system for investigating spectral tuning in microbial rhodopsins since both share >78% of their amino acid residues (200 out of 247 identical amino acid residues), while their visible absorption peaks differ by almost 40 nm.  In contrast, the four types of H. salinarum rhodopsins show a comparable variation in absorption maxima, but share only 25-50% of their residues.

Using structural modeling comparisons and mutagenesis, we identify a single amino acid residue at position 105 that functions as a spectral tuning switch and accounts for most of the spectral difference between the two pigment families.  In addition, site directed mutagenesis of Green-PR- position 105 to several different amino acids; reveal that different amino acids at position 105 cause direct effects on the absorption spectra and photocycle rate.  These results indicate that position 105 acts as a switch for both spectral and photochemical properties of PR.

Furthermore, looking at natural environments, we found novel PR gene clusters spanning the range of 540-505 nm and containing changes in the same identified key switch residue leading to changes in their absorption maxima.  We characterize one natural variant from the Red Sea (RED29) as an example for spectral tuning mechanism in natural marine environments.  Our observations demonstrate that position 105 single-residue switch mechanism is also the major determinant of proteorhodopsin wavelength regulation in natural marine environments.