Correlation of the O-Intermediate Rate with the pKa of Asp-75 in the Dark, the Counterion of the Schiff Base of Pharaonis Phoborhodopsin (Sensory Rhodopsin II)

Abstract

Pharaonis phoborhodopsin (ppR), also called pharaonis sensory rhodopsin II, NpSRII, is a photoreceptor of negative phototaxis in Natronomonas (Natronobacterium) pharaonis. The photocycle rate of ppR is slow compared to that of bacteriorhodopsin, despite the similarity in their x-ray structures. The decreased rate of the photocycle of ppR is a result of the longer lifetime of later photo-intermediates such as M- (ppR(M)) and O-intermediates (ppR(O)). In this study, mutants were prepared in which mutated residues were located on the extracellular surface (P182, P183, and V194) and near the Schiff base (T204) including single, triple (P182S/P183E/V194T), and quadruple mutants. The decay of ppR(O) of the triple mutant was accelerated approximately 20-times from 690 ms for the wild-type to 36 ms. Additional mutation resulting in a triple mutant at the 204th position such as T204C or T204S further decreased the decay half-time to 6.6 or 8 ms, almost equal to that of bacteriorhodopsin. The decay half-times of the ppR(O) of mutants (11 species) and those of the wild-type were well-correlated with the pK(a) value of Asp-75 in the dark for the respective mutants as spectroscopically estimated, although there are some exceptions. The implications of these observations are discussed in detail.

All-Optical Switching in Pharaonis Phoborhodopsin Protein Molecules

“…Recently, new ppR mutants (T204C, T204S, S/E/T/A, S/E/T/C, S/E/T/S, etc.) with azide have been reported which exhibit the pR and pR state decays ms, respectively, which is comparable to that in bR [29]. Hence, ppR can provide switching time of the same order as bR.…”

“…The rate constants of the intermediates, especially pR and pR states can be tailored by various techniques [25]- [29]. For instance, addition of azide affects the pR state life time and replacing the amino acid (Val-108) by mathionine (Val-108 mutant of ppR) causes three times faster decay of pR state and removes the shoulder of its spectrum [25]- [29].…”

“…Since the kinetic and spectral properties of ppR can also be tailored by physical, chemical, and genetic engineering techniques, the switching characteristics can be optimized to increase the contrast and obtain faster response time [20]- [29]. Recently, new ppR mutants (T204C, T204S, S/E/T/A, S/E/T/C, S/E/T/S, etc.)…”

Steric Constraint in the Primary Photoproduct of an Archaeal Rhodopsin from Regiospecific Perturbation of C−D Stretching Vibration of the Retinyl Chromophore

“…The wild-type and mutant proteins of SRII from Natronomonas pharaonis were prepared as described previously (15)(16)(17). Briefly, the SRII protein with a histidine tag at the C-terminus was expressed in Escherichia coli, solubilized with 1.5% n-dodecyl -Dmaltoside, and purified by Ni column chromatography.…”

Functional Importance of the Interhelical Hydrogen Bond between Thr204 and Tyr174 of Sensory Rhodopsin II and Its Alteration during the Signaling Process

“…This plasmid encodes six histidines in the C terminus, and it was named pYS001. Plasmids of T204A, T204S, T204C, and Y174F without HtrII were constructed as previously described (17,21). For the preparation of T204C-HtrII, T204A-HtrII, T204S-HtrII, and Y174F-HtrII expression plasmid, the 5Ј-and 3Ј-ends of the mutant genes were mutated by PCR to get NcoI and SpeI restriction sites, respectively.…”