Yukinori Taniguchi

Application of HaloTag Protein to Covalent Immobilization of Recombinant Proteins for Single Molecule Force Spectroscopy

We have developed the HaloTag system for the covalent immobilization of polyproteins onto a mica substrate for single molecule force spectroscopy using the atomic force microscope. A recombinant fusion polyprotein of titin I27 with HaloTag7 protein was produced, and the covalent and site-specific attachment on a HaloTag-ligand-modified mica surface was confirmed by force-extension measurements. Two mechanical unfolding intermediates of HaloTag7 protein were found by contour length analysis. This tethering method allows site-specific covalent immobilization of a protein that complements the standard method utilizing thiol-gold interaction, thus facilitating force-extension measurements for cysteine-containing proteins.

Application of fluorescence resonance energy transfer (FRET) to investigation of light-induced conformational changes of the phoborhodopsin/transducer complex

The photoreceptor phoborhodopsin (ppR; also called sensory rhodopsin II) forms a complex with its cognate the Halobacterial transducer II (pHtrII) in the membrane, through which changes in the environmental light conditions are transmitted to the cytoplasm in Natronomonas pharaonis to evoke negative phototaxis. We have applied a fluorescence resonance energy transfer (FRET)‐based method for investigation of the light‐induced conformational changes of the ppR/pHtrII complex. Several far‐red dyes were examined as possible fluorescence donors or acceptors because of the absence of the spectral overlap of these dyes with all the photointermediates of ppR. The flash‐induced changes of distances between the donor and an acceptor linked to cysteine residues which were genetically introduced at given positions in pHtrII(1–159) and ppR were determined from FRET efficiency changes. The dye‐labeled complex was studied as solubilized in 0.1%n‐dodecyl‐β‐d‐maltoside (DDM). The FRET‐derived changes in distances from V78 and A79 in pHtrII to V185 in ppR were consistent with the crystal structure data (Moukhametzianov, R. et al. [2006] Nature, 440, 115–119). The distance from D102 in pHtrII linker region to V185 in ppR increased by 0.33 Å upon the flash excitation. These changes arose within 70 ms (the dead time of instrument) and decayed with a rate of 1.1 ± 0.2 s. Thus, sub‐angstrom‐scale distance changes in the ppR/pHtrII complex were detected with this FRET‐based method using far‐red fluorescent dyes; this method should be a valuable tool to investigate conformation changes in the transducer, in particular its dynamics.