Comprehensive observations in structural and functional dynamics of biomolecular systems, which can express higher hierarchic functions by the assembly of proteins or their domains, are crucial to reveal their molecular mechanism. ABC transporters are one of the typical biomolecular systems, which can transport the allocrites through their channel in the transmembrane domain (TMD) along the binding and hydrolysis of ATP in the cytosolic nucleotide-binding domain (NBD). Here, we focused to observe the allocrite transport, ATP hydrolysis, and conformational changes in TMD, and align them along the reaction axis. The transport could be observable by selecting heme ABC transporters, because heme absorb the UV/visible light and it changes the spectrum along its location or ligand. A heme importer, BhuUV-T, was selected for the target, whose structure has been clarified by X-ray crystallography.
The time-resolved (TR) spectroscopy was implemented by combining the microfluidic mixer with several micro-spectroscopy, in which the reaction was triggered by the diffusional mixing of ATP to BhuUV-T/heme complex. TR UV/visible absorption spectroscopy clarified the increase of the heme bound with DDM micelle with the time constant of 100 ms after the mixing, suggesting that the heme release from the complex to the DDM micelle in solvent occur with the time constant of 100 ms. ATP hydrolysis was monitored by the TR FT-IR spectrum with bright infrared beam in BL43IR/SPring-8. The reaction rates of ATP hydrolysis were identical to that of heme transport in absorption spectroscopy within the error, indicating that the allocrite transport is coupled with ATP hydrolysis.
Intermediate states during ATP binding and hydrolysis were also monitored by trapping with AMP-PNP, ADP/VO4, and ADP. UV/visible absorption spectroscopy revealed that the heme is releasee with ADP/VO4 and ATP, but not with AMP-PNP, which indicate that the release of the heme is triggere by the ATP hydrolysis as suggested in TR measurements. Environmental changes of the specific sites and conformational changes of TMD during the ATP reactions were investigated by CW-ESR and pulse-ESR, respectively, for the spin-labeled BhuUV-T. Based on these spectroscopic measurements, the detailed mechanism of biomolecular systems will be discussed.