Use of Dynamic Light Scattering to Detect the Growth of Amyloid Fibrils in HEWL.

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Use of Dynamic Light Scattering to Detect the
Growth of Amyloid Fibrils in HEWL.
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What are they? Why should we study them?

• Proteins bonded in a very specific manner
• How they are formed is not understood
• Associated with many diseases, including
• Alzheimers Disease
• Mad Cow Disease

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Hen Egg White Lysozyme

• In water, HEWL is in folded state
• Hydrogen bonding
• TFE partially unfolds, then stabilizes HEWL
• Newly exposed portions of HEWL should bond to
  each other, forming strands

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How Could We Study Them?

• We wanted to characterize their rate of formation
• Disturbing the proteins would make any data
  collected questionable
• How could we solve this problem?

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Dynamic Light Scattering!
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What is DLS?

• Detects the changing interference pattern of
  laser light scattered by small particles in
  solution.
• From the rate of change, we can measure the
  diffusion rate and size of the particles.

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(No Transcript)
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Light Scattering in Action!
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What is the Autocorrelation Function?

• Put simply the average of the time varying
  portion of the intensity at some initial time, t,
  with the time varying portion of the intensity at
  some later time, t?t.

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OK, It isnt really that simple

• This calculation is done for many values of ?t.
• It is repeated many times (in our case, 400),
  each time averaging the new result with the
  average of all the previous results.

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Why would you do that?

• For small values of ?t, the signal is still
  correlated with the signal at t 0
  positive average intensity.
• For large values of ?t, this is not true, and the
  autocorrelation function will eventually average
  out to zero.

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Key Point

• By measuring how long it takes the function to go
  to zero, we can tell how fast the particles are
  moving!

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For All You Visual Learners
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Microsphere Autocorrelation Function, t 3.89 ms.
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Single Exponential Autocorrelation Function
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Multiple Exponential Autocorrelation Function
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Multiple exponential fit
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Evolution of the Autocorrelation Function
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Time Constants Vs. Incubation Time
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In Conclusion

• Succeeded in producing a DLS setup
• Induced aggregation in HEWL with TFE
• Unable to prove that aggregate contained Amyloid
  Fibrils
• With more time, experiment could be completed

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Special Thanks

• Dr. Stephen Hagen
• Dr. Robert DeSerio
• Caleb Carswell
• University of Florida Physics Department

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(Applause)