title: Mapping of the local environmental changes in proteins by cysteine scanning.
abstract: Protein conformational changes, which regulate the activity of proteins, are induced by the alternation of intramolecular interactions. Therefore, the detection of the local environmental changes around the key amino acid residues is essential to understand the activation mechanisms of functional proteins. Here we developed the methods to scan the local environmental changes using the vibrational band of cysteine S-H group. We validated the sensitivity of this method using bathorhodopsin, a photoproduct of rhodopsin trapped at liquid nitrogen temperature, which undergoes little conformational changes from the dark state as shown by the X-ray crystallography. The cysteine residues were individually introduced into 15 positions of Helix III, which contains several key amino acid residues for the light-induced conformational changes of rhodopsin. The shifts of S-H stretching modes of these cysteine residues and native cysteine residues upon the formation of bathorhodopsin were measured by Fourier transform infrared spectroscopy. While most of cysteine residues demonstrated no shift of S-H stretching mode, cysteine residues introduced at positions 117, 118, and 122, which are in the vicinity of the chromophore, demonstrated the significant changes. The current results are consistent with the crystal structure of bathorhodopsin, implying that the cysteine scanning is sensitive enough to detect the tiny conformational changes.
authors: Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan; Graduate School of Materials Science, Nara Institute of Science and Technology, Nara 630-0192, Japan.
authors: Yoichi
authors: Y
authors: Yamazaki
authors: Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan.
authors: Tomoko
authors: T
authors: Nagata
authors: Department of Biology and Geosciences, Graduate School of Science, Osaka City University, Osaka 558-8585, Japan.
authors: Akihisa
authors: A
authors: Terakita
authors: Department of Materials Science and Engineering, Nagoya Institute of Technology, Nagoya 466-8555, Japan.
authors: Hideki
authors: H
authors: Kandori
authors: Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan.
authors: Yoshinori
authors: Y
authors: Shichida
authors: Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan.
authors: Yasushi
authors: Y
authors: Imamoto
keywords: bathorhodopsin; conformational change; isomerization; rhodopsin; vibrational spectroscopy