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Kwang Hwan Jung

Advisor: John Spudich, Ph.D.

The molecular complex containing the seven transmembrane helix phototaxis receptor Sensory Rhodopsin I (SRI) and transducer protein HtrI (Halobacterial Transducer for SRI) mediates color-sensitive phototaxis responses in the archaeon Halobacterium salinarum. Orange light causes an attractant response by a one-photon reaction and white light (orange + UV light) a repellent response by a two-photon reaction. Three aspects of SRI-HtrI structure/function of signal transduction pathway were explored. First, the coupling of HtrI to the photoactive site of SRI was analyzed by mutagenesis and kinetic spectroscopy. Second, SRI-HtrI mutations and suppressors were selected and characterized to elucidate the color-sensing mechanism. Third, signal relay through the transducer-bound histidine kinase was analyzed using an in vitro reconstitution system with known and newly identified taxis components.

21 mutations on HtrI were introduced by site-directed mutagenesis. Several replacements of charged residues perturbed the photochemical kinetics of SRI which led to the finding of a cluster of residues at the membrane/cytoplasm interface in HtrI electrostatically coupled to the photoactive site of SRI. We found by laser-flash kinetic spectroscopy that the transducer and these residues have specific effects on the light-induced proton transfer between the retinal chromophore and the protein. The rates of proton transfer indicate that HtrI effects the electrostatic environment of the protonated Schiff base. HtrI not only receives the signal from SRI but also optimizes the photochemical reaction process for SRI signaling.

One of the mutations showed an unusual mutant phenotype we called “inverted” signaling, in which the cell produces a repellent response to normally attractant light. Therefore, this mutant (E56Q of HtrI) had lost the color-discrimination by the SRI-HtrI complex. We used suppressor analysis to better understand the phenotype. 15 distinct intragenic and extragenic suppressor mutants were isolated from orange-light inverted signaling mutants by random PCR mutagenesis and a capillary migration method. The locations of the suppressors indicated that the cytoplasmic end of transmembrane helix-2 and the initial part of the cytoplasmic domain of HtrI and SRI are crucial for coupling of the two membrane proteins. Certain suppressors resulted in return of attractant responses to orange light but with inversion of the normally repellent response to white light to an attractant response. To explain this and other results, we formulated the Conformational Shuttling model in which the HtrI-SRI complex is poised in a metastable equilibrium of two conformations shifted in opposite directions by orange and white light. The mutations affect the Keq of the 2-conformational equilibrium controlling flagellar motor switching. We tested this model by behavioral analysis (computerized cell tracking and motion study) of double mutants of inverting and suppressing mutations and the results confirmed the equilibrium-shift explanation. Furthermore, support for the model comes from pH and temperature effects on the behavioral responses.

We developed an in vitro system for measuring the effect of purified transducer on the histidine-kinase CheAH that controls the flagellar motor switch. The rate of kinase autophosphorylation was stimulated >2 fold in the reconstitution of the complete signal transduction system from purified components (HtrXI, CheWH, CheAH, and CheYH) from H. salinarum. The in vitro assay also showed that the kinase activity was reduced in the absence and in the presence of high levels of linker protein CheWH, as expected from its putative role as a coupling protein with separate transducer and kinase binding sites.

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Research Info

Protein-Protein Interaction Between Phototaxis Receptor Sensory Rhodopsin I and Its Transducer HtrI