Resonance Raman observation of the structural dynamics of FixL on signal transduction and ligand discrimination.
FixL is a heme-based O2 sensor protein, which responds to low O2 concentrations by activating the transcriptional activator FixJ. Signal transduction is initiated by the dissociation of O2 from the sensor domain of FixL, resulting in protein conformational changes that are transmitted to a histidine kinase domain. To gain insight into the FixL sensing mechanism, we monitored changes in the protein's structure in the picosecond to millisecond time frame, following the dissociation of the ligand using time-resolved resonance Raman spectroscopy. This study presents the time-resolved resonance Raman spectra of Sinorhizobium meliloti FixL upon O2 dissociation, as well as upon CO dissociation. The FixL spectra show that there are three steps in the dynamic structural changes that result from ligand dissociation. Ligand-dependent structural dynamics are observed in the earliest step. On the basis of comparisons of these structural changes, a scheme for the signal transduction of FixL is proposed which supports the FG loop switch mechanism. Similar spectral changes were observed both for the sensor domain and for the full-length protein, although structural changes occurred faster with the former than with the latter. This difference in rate suggests that the structural changes occurring in the heme pocket are coupled to those of the kinase domain. The implications of these results for FixL's sensing mechanism are discussed.
Hiruma
Yusuke
Y
Graduate School of Science and Technology, Kobe University, Nada, Kobe 657-8501, Japan.
Kikuchi
Akihiro
A
Tanaka
Atsunari
A
Shiro
Yoshitsugu
Y
Mizutani
Yasuhisa
Y
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D002248:Carbon Monoxide / Q000378:metabolism; D006418:Heme / Q000737:chemistry; D006420:Hemeproteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D000071677:Histidine Kinase; D008024:Ligands; D010100:Oxygen / Q000378:metabolism; D011485:Protein Binding; D047428:Protein Kinase Inhibitors / Q000737:chemistry; D011494:Protein Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D015398:Signal Transduction / Q000502:physiology*; D016962:Sinorhizobium meliloti / Q000737:chemistry / Q000502:physiology; D013059:Spectrum Analysis, Raman; D013816:Thermodynamics
D001426:Bacterial Proteins; D006420:Hemeproteins; D008024:Ligands; D047428:Protein Kinase Inhibitors; D006418:Heme; D002248:Carbon Monoxide; D011494:Protein Kinases; C067929:FixL protein, Bacteria; D000071677:Histidine Kinase; D010100:Oxygen