Unfolding RXRalpha

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Unfolding RXRalpha
Students Nancy Doremus and Jessie
Dodge-Gittins Mentors Zeb Jones and Mike Stoops
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Background Information

• The way a protein is assembled and folded is
  very important in determining its purpose. Its
  like a second genetic code.
• RXRalpha is a receptor protein for retenoid
  (vitamin A derivatives) molecules.
• These receptors are implicated in cell
  differentiation, mammalian development, and skin
  diseases like acne and psoriasis.

Jessie weighing buffer materials
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Purpose

• Understand the stability of RXRalpha
• Comprehend dynamics of the protein unfolding and
  folding
• Fit data to a theoretical folding model

Jessie and Nancy getting ice
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Process

• Purifying the RXRalpha protein
• Checking for contamination
• Testing concentration
• Collecting data after exposure to a chemical
  denaturant

Jessie using the pipette
Nancy filling test tubes
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Centrifuge
The RXRalpha sample needed a higher concentration
for the experiment, so the centrifuge was used to
purify it. The centrifuge spun the protein at
4000 RPMs for 40 minutes, which separated the
protein from the filtrate. After testing the
concentration of the protein, it was found that
it wasnt high enough, so the centrifuge was
needed again.
Looking down into the centrifuge
Nancy and Jessie loading the centrifuge
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Bradford Protein Assay
The spectrophotometer measures the absorbency of
light through a sample.
It was used to find the concentration of the
RXRalpha protein.
Samples of known concentration ( Bovine )
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Electrophoresis
Electrophoresis gives a rough molecular weight
range. SDS ( Sodium dodecyl sulfate ) in the
gel acts as a surfactant. Surfactant denatures
proteins allowing for band resolution as proteins
migrate through an electric field. Multiple
bands suggest contamination.
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Fluorometer
The fluorometer shines light through a sample
which makes some amino acids fluoresce. The
machine detects this and sends the data to a
computer where it is compiled into a graph.
A denaturant ( guanadine ) was added to the
RXRalpha sample to divide the tetramers into
monomers. This change is visible on the graphs.
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Statistical Analysis

• Import data into Excel
• Calculate net fluorescence (subtracting buffer
  blank)
• Calculate weighted mean wavelength lt ? gt

lt ? gt S ( I ? ) / S ( I ) ? wavelength
I net fluorescence

• Correlate calculated lt ? gt to known values.

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Conclusions
Weighted-mean average wavelengths correspond well
with previous data. Each protein configuration
has a concise average wavelength, distinguishable
by several nanometers. The unfolding scheme
appears to follow a progression from tetramer to
dimer and finally, as the chemical denaturant
(guanadine) concentration approached 5.5 M,
monomer. Further experiments might assay the
refolding of the protein as guanadine is diluted.
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Electrophoresis Standards