Microsecond atomic force sensing of protein conformational dynamics: Implications for the primary light-induced events of bacteriorhodopsin

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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

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Are protein conformational change and charge
redistribution related?

• The leading hypothesis is that protein
  conformational change is initiated through
  isomerization around the C13C14 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.

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Atomic Force Sensing
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AFS detects conformational change of bR on
microsecond scale

• After exciting membrane with 532 nm light, AFS
  data indicates expansion of membrane.

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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.

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AFS data suggests redistribution of charge in
chromophore upon excitation
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AFS detects changes not observable by absorption
spectroscopy

• Data from AFS measurements indicate
  conformational changes even in molecules
  incapable of undergoing C13C14 isomerization.
• Perhaps the conformational changes detected
  through AFS become physiologically relevant only
  if isomerization is possible.

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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.

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Questions ?