TOWARDS THE EXPERIMENTAL VALIDATION OF A NEW MEMBRANE PROTEIN FOLDING MODEL: A report on my work in Dr.Judith Klein- Seetharaman

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TOWARDS THE EXPERIMENTAL VALIDATION OF A NEW
MEMBRANE PROTEIN FOLDING MODELA report on my
work in Dr.Judith Klein- Seetharamans lab from
1st September 2005 to 31st December 2005

• Varsha Shridhar

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The Two-stage Hypothesis for the Folding of
Membrane Proteins

• Step 1- The transmembrane segments of membrane
  proteins form helices independently in the lipid
  environment
• Step 2- Individual helices make helix-helix
  contacts and come together to assemble the whole
  protein.
• Protein folding studies on bacteriorhodopsin
  strongly support this model. Bacteriorhodopsin
  helix fragments can associate in vitro to form
  the whole protein.

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• Studies on rhodopsin, however, do not support
  this model. Instead, they seem to indicate that
  tertiary contacts between residues in the loops
  connecting the helices and those in the helices
  themselves are important in the assembly of
  rhodopsin.

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• Thus, it is thought that, in the case of
  rhodopsin at least, long range interactions
  between loop and transmembrane residues are more
  important than the short range interactions
  between transmembrane helices.
• It is even possible that these long range
  interactions take place first, and somehow
  cooperatively induce the correct folding of the
  protein.
• The likely folding core of rhodopsin has been
  predicted by various simulation methods- FIRST
  and Gaussian Network Modelling.

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Approaches Planned to Prove the Long-Range
Interactions Model

• A) Investigate the stability of rhodopsin folding
  core mutants
• B) Label cysteines inside and outside of the
  predicted folding core and attach biophysical
  probes to them, such as 19F labels for NMR
  structural studies of the folding core

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What I did during the past 4 months

• 1) A preliminary rhodopsin (Rho) stability assay
• 2) Purification of Rhodopsin and Labeling with
  different reagents (19F, TET and PDS)
• 3) Hydroxylamine assay

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Why I did what I did

• 1) The Rho stability assay Measures of Rho
  stabilitya) The number of exposed cysteines on
  rhodopsin
• b) The proteins resistance to denaturing agents.
• Thus, a) Find out the least SDS required to
  denature w/t Rho. Compare it with that required
  to denature mutant Rho.This could give an early
  idea about the relative stabilities of the two.
• b) We could also do the same by finding out the
  number of Cys labeled on a Rho sample treated
  with a given concentration of SDS.

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Results of this experiment- 0.065 SDS is the
least concentration reqd. to completely denature
w/t Rho
Rhodopsin is denatured by SDS. This is
spectroscopically seen with the shifting of the
500nm peak of wildtype rhodopsin to 440nm
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Kinetics of Denaturation can be studied by
Cysteine Labeling
Cysteine labeling in wildtype rhodopsin. By 60
minutes after addition of PDS, both the outer
cysteines are labeled in wildtype rhodopsin.
Cycles refer to 5 minute intervals
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0.1 SDS is added at cycle 30.
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Cysteine labeling in rhodopsin treated with 0.1
SDS. Each cycle refers to a time span of 5 minutes
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• 2) Purification of Rho from bovine retinae
• 1 mg
• 200ug 600ug 200ug
• Unlabeled NEM labeled
  Unlabeled
• BV 250uL 500uL
  250uL
• 1.5 mL eppendorf BioRad Poly
  5mL falcons Prep
  2mL
  Affinity Chromatography column
  

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• 19F-labeled Rho using TET

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The Hydroxylamine Story

• Previous studies had shown that cysteines that
  are inaccessible in the dark state of rhodopsin
  become accessible upon light activation and
  leaving of the retinal.
• We would like to make use of this fact and label
  these cysteines with biophysical probes

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• Rhodopsin is not very stable after
  light-activation.
• Hence, we want to remove the retinal faster than
  through natural decay (to prevent aggregation of
  Rho)
• Hydroxylamine reacts with the retinal-Lys296
  Schiff base releasing retinaloxime, which readily
  leaves the binding pocket
• It is crucial, however, to remove hydroxylamine
  efficiently during the rhodopsin preparation
  protocol, as it tends to react with the cysteine
  labels.

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• I spent the remainder of my rotation on
  establishing an assay to detect hydroxylamine.
• I obtained preliminary evidence suggesting that
  the reaction between hydroxylamine, cystine and
  PDS can be used as an assay.

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• Assays tried
• A) With PDS

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• With dTT

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• With Cysteine

Absorbance spectra of sample containing 150uM
PDS, 70mM hydroxylamine and 0.5uM Cysteine.
Cycles refer to spectra taken at 10-minute
intervals.
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• With Fe3 and Sodium SalicylateFe3 reacts with
  salicylate to form a colored complexFe3 (aq)
  C7H5O3- (aq) Fe(C7H5O3)2 (aq)
• Hydroxylamine would reduce Fe3 to Fe2, making
  this latter species unreactive with salicylate.
  Adding an excess of Fe3 to a sample containing
  an unknown concentration of hydroxylamine would
  convert some of the Fe3 to Fe2. The remaining
  Fe3 could then react with salicylate. The
  concentration of the product formed could then be
  checked by absorption spectroscopy, and the
  amount of hydroxylamine back-calculated

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The graphs above show that the reaction between
Ferric Nitrate and Sodium Salicylate is perfectly
stoichiometric. The absorbance maximas (at 323
nm) lie in a straight line which passes through
the origin.
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As time increases, more Fe3 is reduced to Fe2
than is predicted by the chemical equation. This
leads to lower and lower absorption maximas.
Low sensitivity of test. Na.Sal refers to sodium
salicylate. The concentration of Na.Sal in the
first three samples is 100uM, and 1mM in the
last.
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• Using NAD Hydroxylamine was expected to reduce
  NAD to give NADH, both of which have distinctive
  spectra

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• Cystine We then studied if hydroxylamine could
  perhaps reduce the disulfide bond in cystine,
  releasing two cysteines, which could then react
  with the PDS .

10uM Cystine is reacted with 0, 1, 5 and 10uM
hydroxylamine. 50uM Cystine is reacted with 50uM
of hydroxylamine. PDS 150uM
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And, even better.
The reaction between 50uM Cystine and 50uM
hydroxylamine does not change with time.
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• A Final Recap
• 1) The major part of my work consisted of
  attempting to find a sensitive assay for
  hydroxylamine. The cystine assay seems now to be
  the most promising.
• 2) Purification of rhodopsin protein was also
  carried out. Unlabelled rhodopsin,rhodopsin
  labeled with NEM (to block the two free cysteines
  on the extracellular domain of the protein) and
  rhodopsin labeled with 19F were prepared
  according to standard protocols, for use in
  future experiments.

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• 3) A smaller experiment carried out with SDS
  attempted to show the results of mutation on the
  stability of the protein, specifically on its
  denaturation kinetics. This study is incomplete.
  For the moment, it just shows that the minimum
  amount of SDS required to completely denature
  wildtype rhodopsin is 0.065.
• This is important in the future NMR based
  experiments where addition of the right amount of
  denaturant will be important to denature the
  protein without contributing too much background
  noise.

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• Acknowledgements

Judith Naveena David Harpreet Fernanda Kalyan Hu
ssein Madhavi
http//www.cs.cmu.edu/naveena/baby
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