Accepted post-doctoral position at Baylor College of Medicine after receiving PhD
Now employed as a Research Associate at Baylor College of Medicine, Houston.
The molecular complex of sensory rhodopsin I (SRI) and its transducer HtrI mediate color-sensitive phototaxis in the archaeon Halobacterium salinarum. Orange light causes an attractant response by a one-photon reaction and white light (orange + UV light) causes a repellent response by a two-photon reaction. Three aspects of this molecular complex were explored: (i) We determined the stoichiometry of SRI and HtrI to be 2:2 by gene fusion analysis. (ii) Cytoplasmic channel closure of SRI by HtrI, an important aspect of their interaction, was investigated by incremental HtrI truncation. (iii) We developed a procedure for reconstituting free SRI and SRI/HtrI complex into liposomes in a manner that reproduces the key interactions we established in native membranes. The proteoliposomes permit conformational changes of SRI induced by light to be detected by EPR and fluorescence probes.
A fusion protein in which the C-terminus of H. salinarum sensory rhodopsin I (SRI) is connected by a flexible linker to the N-terminus of its transducer I (HtrI) was constructed and expressed in H. salinarum. The fusion protein mediated 1-photon and 2-photon phototaxis responses comparable to those produced by the wild-type complex. Immunoblot analysis 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. Measurement of the photochemical reaction kinetics and pH titration of the absorption spectra established that both SRI domains are complexed to HtrI in the fusion protein, and therefore the stoichiometry is 2:2.
HtrI-binding to SRI closes a cytoplasmic proton-conducting channel in SRI that opens during the free SRI photochemical reaction cycle. To investigate the channel closure, a series of HtrI mutants truncated in the membrane-proximal cytoplasmic portion of an SRI-HtrI fusion were constructed. We found that binding of the membrane-embedded portion of HtrI is insufficient for channel closure, whereas cytoplasmic extension of the second HtrI transmembrane helix by 13 residues blocks proton conduction through the channel as well as full-length HtrI. The closure activity is localized to 5 specific residues, each of which incrementally contributes to reduction of proton conductivity. Moreover, these same residues in the dark incrementally and proportionally increase the pKa of the Asp76 counterion to the protonated Schiff base chromophore in the membrane-embedded photoactive site. We conclude that this critical region of HtrI alters the dark conformation of SRI as well as light-induced channel opening. The 5 residues in HtrI correspond in position to 5 residues demonstrated on the homologous HtrII to interact with the E-F loop of its cognate receptor SRII (Yang et al., JBC, in press). These results support a model in which the membrane-proximal region of Htr proteins interact with their cognate receptors’ cytoplasmic E-F loops as part of the signal-relay coupling between the proteins.
We developed a procedure for reconstituting free SRI and truncated SRI-HtrI fusion into liposomes. The free SRI and SRI-HtrI complex exhibit photocycles with opened and closed cytoplasmic channels, respectively, as in the membrane. This opens the way for study of the light-induced conformational change and the interaction in vitro by fluorescence and spin-labeling. Single-cysteine mutations were introduced into helix F of SRI, labeled with a nitroxide spin probe and a fluorescence probe, and reconstituted into proteoliposomes. Light-induced conformational changes were detected by fluorescence changes in the complex. The kinetics of fluorescence change matched the rise and decay of the intermediate of the SRI photocycle formed by proton transfer from the chromophore to the Asp76 acceptor on helix C. We also succeeded in labeling SRI molecule with a nitroxide spin probe in vitro and got clear EPR spectra. The fluorescence change and EPR signals can now be used as the readout of signaling to analyze mutants and the kinetics of signal relay.
Signal Relay between Two Integral Membrane Proteins: the Sensory Rhodopsin Transducer Molecular Complex