Demonstration of 2:2 Stoichiometry in the Functional SRI−HtrI Signaling Complex in Halobacterium Membranes by Gene Fusion Analysis

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Abstract
A fusion protein in which the C-terminus of Halobacterium salinarum sensory rhodopsin I (SRI) is connected by a flexible linker to the N-terminus of its transducer (HtrI) was constructed and expressed in H.salinarum. The fusion protein mediated attractant responses to orange light and repellent responses to UV/violet light that were comparable to those produced by the wild-type SRI−HtrI complex. Immunoblot analysis of H.salinarum membrane proteins demonstrated intact fusion protein and no detectable proteolytic cleavage products. Rapid oxidative cross-linking of a monocysteine mutant in the HtrI domain confirmed that the fusion protein exists as a homodimer in the membrane. HtrI-free SRI and HtrI-complexed SRI have been shown previously to exhibit large differences in the pH dependence of their photocycle kinetics and in the pKa of Asp76 that controls a pH-dependent spectral transition in SRI. These differences were used to assess whether only one or both SRI domains in the fusion protein were complexed properly to the HtrI homodimer. Measurement of the photochemical activity, the photocycle kinetics, and the absorption spectra at various pH values established that both SRI domains are complexed to HtrI in the fusion protein, and therefore the stoichiometry is 2:2. Closer examination of the HtrI effect on SRI revealed that Asp76 titration in HtrI-free SRI fits two pKa values, with 98% and 2% of the molecules titrating with pKa's of 7 and 9, respectively. The same two pKa's of Asp76 are evident in HtrI-complexed SRI, but with 13% with pKa of 7 and 87% with pKa of 9 and a similar bias toward the pKa of 9 in the fusion protein. Titration of the fusion protein with Ala substitution at Arg73, a residue in the photoactive site, in the SRI domain indicates that a basic residue at Arg73 is necessary for the lower pKa to be observed. A model in which Arg73 plays a role in the HtrI effect on SRI is discussed.