Photochromic bacteriorhodopsin mutant with high holographic efficiency and enhanced stability via a putative self-repair mechanism.
The Q photoproduct of bacteriorhodopsin (BR) is the basis of several biophotonic technologies that employ BR as the photoactive element. Several blue BR (bBR) mutants, generated by using directed evolution, were investigated with respect to the photochemical formation of the Q state. We report here a new bBR mutant, D85E/D96Q, which is capable of efficiently converting the entire sample to and from the Q photoproduct. At pH 8.5, where Q formation is optimal, the Q photoproduct requires 65 kJ mol(-1) of amber light irradiation (590 nm) for formation and 5 kJ mol(-1) of blue light (450 nm) for reversion, respectively. The melting temperature of the resting state and Q photoproduct, measured via differential scanning calorimetry, is observed at 100 °C and 89 °C at pH 8.5 or 91 °C and 82 °C at pH 9.5, respectively. We hypothesize that the protein stability of D85E/D96Q compared to other blue mutants is associated with a rapid equilibrium between the blue form E85(H) and the purple form E85(-) of the protein, the latter providing enhanced structural stability. Additionally, the protein is shown to be stable and functional when suspended in an acrylamide matrix at alkaline pH. Real-time photoconversion to and from the Q state is also demonstrated with the immobilized protein. Finally, the holographic efficiency of an ideal thin film using the Q state of D85E/D96Q is calculated to be 16.7%, which is significantly better than that provided by native BR (6-8%) and presents the highest efficiency of any BR mutant to date.
Department of Molecular and Cell Biology, University of Connecticut , 91 North Eagleville Road, Storrs, Connecticut 06269, United States.
Matthew J
MJ
Ranaghan
Jordan A
JA
Greco
Nicole L
NL
Wagner
Rickinder
R
Grewal
Rekha
R
Rangarajan
Jeremy F
JF
Koscielecki
Kevin J
KJ
Wise
Robert R
RR
Birge
D001436:Bacteriorhodopsins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D002152:Calorimetry, Differential Scanning; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D013056:Spectrophotometry, Ultraviolet
D001436:Bacteriorhodopsins