Room temperature trapping of rhodopsin photointermediates.
By suspending bovine rhodopsin in trehalose-water glass films, it is possible to trap photostates in the light-activation process. Because of the unusually high vitrification temperature of trehalose-water mixtures, this trapping can be accomplished at room temperature. This allows for a facile investigation of the spectroscopic properties of rhodopsin's photointermediates. Depending on experimental conditions, it is possible to trap photolysis products that have visible absorbance spectra closely resembling the two different photointermediates, metarhodopsin I and metarhodopsin II. When rhodopsin is maintained in the native rod outer segment membrane, the photolysis product has the spectral properties of metarhodopsin I. Upon detergent solubilization, the photolysis product closely resembles metarhodopsin II. Ultraviolet circular dichroism spectra show that the metarhodopsin I product had no change in secondary structure compared with unbleached rhodopsin. The metarhodopsin II product did show a significant decrease in alpha-helical content. Resonance energy transfer was measured from extrinsic probes located on each of the cytoplasmic cysteine residues to the retinal in the trapped photoproducts. It is seen that these distances are the same for rhodopsin and metarhodopsin I while metarhodopsin II shows considerably shorter distances. Metarhodopsin II is intimately associated with the signal transduction process, and the present results suggest that large structural changes have occurred in the transition to this state. These results demonstrate the utility of room temperature trapping of photostates in trehalose-water glasses.
Sikora
S
S
Department of Chemistry, University of Denver, Colorado 80208.
Little
A S
AS
Dewey
T G
TG
D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D004735:Energy Transfer; D005456:Fluorescent Dyes; D005898:Glass; D008027:Light; D010777:Photochemistry; D010782:Photolysis; D017433:Protein Structure, Secondary; D012243:Rhodopsin / Q000031:analogs & derivatives / Q000737:chemistry*; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013696:Temperature; D014199:Trehalose; D014867:Water
D005456:Fluorescent Dyes; D014867:Water; C013110:metarhodopsins; D012243:Rhodopsin; D014199:Trehalose