Role of charged residues of pharaonis phoborhodopsin (sensory rhodopsin II) in its interaction with the transducer protein.
pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, NpSRII) is a receptor for negative phototaxis in Natronomonas (Natronobacterium) pharaonis. In membranes, it forms a 2:2 complex with its transducer protein, pHtrII, which transmits light signals into the cytoplasmic space through protein-protein interactions. We previously found that a specific deprotonated carboxyl of ppR or pHtrII strengthens their binding [Sudo, Y., et al. (2002) Biophys. J. 83, 427-432]. In this study we aim to identify this carboxyl group. Since the D75N mutant has only one photointermediate (ppR(O)(-)(like)) whose existence spans the millisecond time range, the analysis of its decay rate is simple. We prepared various D75N mutants such as D75N/D214N, D75N/K157Q/R162Q/R164Q (D75N/3Gln), D75N/D193N, and D75N/D193E, among which only D75N/D193N did not show pH dependence with regard to the ppR(O)(-)(like) decay rate and K(D) value for binding, implying that the carboxyl group in question is from Asp-193. The pK(a) of this group decreased to below 2 when a complex was formed. Therefore, we conclude that Asp-193(p)()(pR) is connected to the distant transducer-ppR binding surface via hydrogen bonds, thereby modulating its pK(a). In addition, we discuss the importance of Arg-162(p)()(pR) with respect to the binding activity.
Sudo
Yuki
Y
Laboratory of Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan.
Iwamoto
Masayuki
M
Shimono
Kazumi
K
Kamo
Naoki
N
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001216:Asparagine / Q000235:genetics / Q000378:metabolism*; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D002338:Carotenoids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D025602:Halorhodopsins; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019633:Natronobacterium / Q000737:chemistry* / Q000235:genetics; D010782:Photolysis; D020283:Photoreceptors, Microbial / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D025603:Sensory Rhodopsins; D015398:Signal Transduction / Q000235:genetics; D055672:Static Electricity
D019843:Archaeal Proteins; D025602:Halorhodopsins; C434451:HtrII protein, Natronobacterium pharaonis; D020283:Photoreceptors, Microbial; D025603:Sensory Rhodopsins; C434450:phototaxis receptor sensory rhodopsin II, Natronobacterium pharaonis; C061508:sensory rhodopsin II protein, archaeal; D001224:Aspartic Acid; D002338:Carotenoids; D001216:Asparagine