Microsecond time-resolved circular dichroism of rhodopsin photointermediates

Area of Science:

• Biochemistry
• Spectroscopy
• Molecular Biology

Background:

• Rhodopsin activation involves transient intermediate states crucial for G protein-coupled receptor (GPCR) signaling.
• Understanding these intermediates provides insights into GPCR function and drug development.
• Previous studies often relied on low-temperature trapping, potentially altering native states.

Purpose of the Study:

• To structurally characterize rhodopsin photointermediates at room temperature using time-resolved circular dichroism (CD).
• To investigate photointermediates with similar absorbance spectra (isochromic) that are challenging to study.
• To elucidate the earliest structural events triggering rhodopsin activation.

Main Methods:

• Time-resolved circular dichroism (CD) spectroscopy from 300 to 700 nm.
• Measurements performed at 5, 100, and 500 microseconds post-photoexcitation.
• Bovine rhodopsin in lauryl maltoside suspension at room temperature.

Main Results:

• CD spectra of lumirhodopsin (5 µs) and metarhodopsin II (500 µs) were consistent with previous low-temperature studies.
• The CD spectrum of lumirhodopsin evolved between 5 and 100 µs, showing reduced rotational strength.
• This spectral change could not be explained by the initial lumirhodopsin CD spectrum alone.

Conclusions:

• The CD spectrum of lumirhodopsin undergoes conformational changes within 5-100 µs.
• These changes are linked to early events like spectral shifts or Schiff base deprotonation/reprotonation.
• Such conformational changes may represent the initial trigger for rhodopsin activation.