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Time-resolved site-directed spin-labeling studies of bacteriorhodopsin: loop-specific conformational changes in M
R Mollaaghababa1, H J Steinhoff, W L Hubbell
1Departments of Biology and Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Biochemistry
|February 2, 2000
Summary
Spin labels in bacteriorhodopsin
Area of Science:
- Biophysics
- Structural Biology
- Protein Dynamics
Background:
- Bacteriorhodopsin undergoes conformational changes during its photocycle.
- Previous studies identified a conformational change during the M to N transition using spin labels.
Purpose of the Study:
- To investigate conformational changes in bacteriorhodopsin using spin labels at various sites.
- To examine the role of the M intermediate in conformational changes, particularly in a D96A mutant.
Main Methods:
- Time-resolved electron paramagnetic resonance (EPR) spectroscopy.
- Site-directed spin labeling in purple membranes.
- Analysis of conformational changes in wild-type and D96A mutant bacteriorhodopsin.
Main Results:
- Only spin labels in the C-D loop detected significant environmental changes after the rise of the M intermediate.
- The D96A mutation prolonged the M intermediate lifetime without altering the conformational change at site 101.
- Conformational changes at site 101 were detected during the M intermediate's prolonged lifetime, even before 410 nm absorbance changes.
Conclusions:
- The C-D loop is a key region for conformational changes during the M intermediate phase.
- The M intermediate population is heterogeneous in real-time.
- Conformational motion at site 101 occurs within the M state, preceding Schiff base reprotonation.