1. Microsecond atomic force sensing of protein conformational dynamics: Implications for the primary light-induced events of bacteriorhodopsin Paper by Rousso et al., PNAS 1997 Presented by Matt Gethers 11/20/08

2. Are protein conformational change and charge redistribution related? The leading hypothesis is that protein conformational change is initiated through isomerization around the C 13 =C 14 bond of the chromophore. Rousso et al. investigated through AFS the hypothesis that light-induced conformational change is initiated through a polarization of the chromophore.

3. Atomic Force Sensing

4. AFS detects conformational change of bR on microsecond scale After exciting membrane with 532 nm light, AFS data indicates expansion of membrane.

5. AFS data agrees with known choromophore specificity and photocycle Exciting membrane with different wavelengths of light shows a peak response ~532 nm. An initial pulse of 532 nm followed by a secondary pulse shows peak response at ~410 nm, the characteristic absorption of the M intermediate.

6. AFS data suggests redistribution of charge in chromophore upon excitation

7. AFS detects changes not observable by absorption spectroscopy Data from AFS measurements indicate conformational changes even in molecules incapable of undergoing C 13 =C 14 isomerization. Perhaps the conformational changes detected through AFS become physiologically relevant only if isomerization is possible.

8. AFS makes possible the ability to sense upstream events Absorption spectroscopy requires all steps in fluorescence to occur to measure activity. AFS offers a means of sensing upstream (biomechanical) steps in the fluorescence pathway – allows us to decompose the process with greater resolution.

9. Questions ?