Demonstration of 2:2 stoichiometry in the functional SRI-HtrI signaling complex in Halobacterium membranes by gene fusion analysis

Biochemistry
Xinpu Chen, J L Spudich

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 pK(a) 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 protei...Continue Reading

Citations

Aug 7, 2002·Current Opinion in Structural Biology·John L Spudich, Hartmut Luecke
Apr 2, 2011·The Journal of Biological Chemistry·Jun SasakiJohn L Spudich
Jun 30, 2009·Journal of Molecular Biology·Daisuke SuzukiYuki Sudo
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