Conformational changes of cytosolic loops of bovine rhodopsin during the transition to metarhodopsin-II: an investigation by Fourier transform infrared difference spectroscopy.
In order to assign the structural changes of the protein, observed in the Fourier transform infrared (FT-IR) difference spectra of the rhodopsin-metarhodopsin-II transition, to specific regions of the protein, rhodopsin was treated by proteases. Nonilluminated and bleached rhodopsin was treated with protease K and papain. Rhodopsin digested in the bleached state was subsequently regenerated with 11-cis-retinal. From these modified samples the rhodopsin-metarhodopsin-II FT-IR difference spectra were measured. Comparing the difference spectra with that of unmodified rhodopsin, clear deviations in the amide-I and amide-II spectral range are observed. This indicates that in the unmodified pigment conformational changes of those parts of the cytosolic surface take place which are susceptible to the proteases. From the larger spectral changes obtained with samples digested in the bleached state it is concluded that the extent of modification is larger. The difference spectra of rhodopsin modified with 10 mM dithiothreitol support the existence of the 4th loop which also undergoes conformational changes. The spectral changes are interpreted in terms of a transition of an ordered structure of the loops in rhodopsin to a more random structure in metarhodopsin-II. The results demonstrate that by combining FT-IR spectroscopy with protein modification by specific proteases, conformational changes of the protein can be localized to specific regions.
Ganter
U M
UM
Institut für Biophysik und Strahlenbiologie, Albert-Ludwig-Universität Freiburg, Germany.
Charitopoulos
T
T
Virmaux
N
N
Siebert
F
F
D000818:Animals; D002417:Cattle; D003600:Cytosol / Q000737:chemistry; D066298:In Vitro Techniques; D010777:Photochemistry; D011487:Protein Conformation / Q000528:radiation effects; D012243:Rhodopsin / Q000031:analogs & derivatives / Q000737:chemistry* / Q000528:radiation effects; D013055:Spectrophotometry, Infrared
C013110:metarhodopsins; D012243:Rhodopsin