Secondary structure of Dna binding peptides

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Secondary structure of Dna binding peptides

• Jeff Samuelson, UCSB Group, Catalyst 2007-2008

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Goals of my project

• Secondary structure of bZip peptide
• Temperature/Structure relationship
• Assumption of structure via micelles
• Efficacy of secondary structure predictors
• Techniques
• Short amino acid chain problems

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GCN4 Protein and bZip Peptide
bZip Amino Acid Sequence N- KDPAALKRARNTEAARRSR
ARKLQRMKQLEAKLAEIE K-C
Splettstoesser, Thomas, Leucine Zipper bound to
DNA
Splettstoesser, Thomas, Leucine Zipper bound to
DNA

• The GCN4 Proteins are transcription factors which
  have a-helical basic structures bonded together
  with a Leucine zipper

• The Basic Leucine Zipper (bZip) is the sequence
  of amino acids which controls the actual DNA
  binding

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Potential Applications of bZip

• As an artificial transcription factor It could
  be used to alter or regulate expression of a gene
• As a method of non-viral gene delivery It could
  be employed to deliver foreign DNA and perform
  tasks relating to gene therapy

Tsutakawa, Susan, Recognition of RNA Polymerase
II and Transcription Bubbles by XPG, CSB, and
TFIIH. Molecular Cell Vol.20, Iss.2
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Protein Analogous Micelles

• bZip needs to retain a-helical secondary
  structure
• Protein Analogous Micelles support DNA
  interaction
• Surrounding peptides are folded into the same
  secondary structures as proteins

Micelles
Proteins
Adapted from R. Tu, Assembly of Peptide
Functionalized Molecular Architectures.
Dissertation, UCSB 2004
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Micelles with bZip
Marullo, Rachel S.
Marullo, Rachel S.

• C16 and di-C16 tails added, force cylindrical
  micelles
• Headgroup a-helical secondary structure like GCN4

hydrophobic tails in core
multivalent a-helical peptide shell
Marullo, Rachel S.
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Testing

• We did testing with Circular Dichroism (CD) in
  the 197-260nm range
• Magnetic field polarizes light which strikes
  sample, then calculate structure based on
  relative absorbance (MRE)

Institute for Storage Ring Facilities, University
of Aarhus
UCSB OLIS RSM circular dichroism spectrometer
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CD Basis Spectra

• The normal Molar Ellipticity (MRE) values for the
  a-helical and random coil structures

Data from Greenfield, Fasman in 1969 journal
Biochemistry
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CD Data Temperature Variation with bZip

• Our original data shows a move to a-helical
  structure which coincides with a drop in
  temperature

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Mathematical Fits

• We used fits from known quantities
• With basis for comparison, structural content was
  approximated

4C
15C
22C
30C
40C
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CD Data Temperature Variation with bZip

• ?-sheet (extended strand) is removed
• No luck with AGADIR, fit to Zimm-Bragg model
• Information only has application as a standard of
  comparison
• Human body temperature is 37C, therefore
  conditions below that are improbable

AGADIR Prediction
?H -4200 J/mol
?S -15.5
Derived from Zimm-Bragg Model of helix-coil
transitions
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Secondary Structure Variation

• CD Spectra at 20C vary greatly between free
  peptide chain and the micelles
• Micelles exhibit near-a structure
• Free peptide chain is very random

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Online Secondary Structure Predictors

• Online structure predictors find the secondary
  structure based on the AA sequence in the PDB
• 75 Accuracy Neural networks with amino acid
  property profile and the position based scoring
  matrix
• 64.4 Accuracy check possible pair frequencies
  within a window of 17 amino acid residues,
  cross-validate on a data base of 267 proteins
• Lower accuracy from cross-referencing with full
  proteins

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Many Thanks

• Thank you to Professor Tirrell, Matt Black, the
  APA, The Camille and Henry Dreyfus Foundation,
  Rena, Ashley, everyone who worked to make this
  possible
• And a very special thank you to Rachel
  Marulloyou have my undying gratitude for
  everything

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Bibliography

• Splettstoesser, Thomas. Leucine Zipper. 2007.
  Splettstoesser, Thomas. Wikipedia. Accessed 26
  June 2008 http//en.wikipedia.org/wiki/ImageLeuci
  ne_zipper.png.
• Tsutakawa, Susan. RNAPII. 2005. Lawrence Berkeley
  National Laboratory, Berkeley. LBL. Accessed 26
  June 2008 http//www.lbl.gov/Science-Articles/Arch
  ive/LSD-TCR.html.
• Tu, R. Assembly of Peptide Functionalized
  Molecular Architectures. 2004. Tu, R. UCSB, Santa
  Barbara
• Brzezinska, Krystyna. CD3. 2007. UCSB, Santa
  Barbara. CD. Accessed 26 June 2008
  http//www.mrl.ucsb.edu/mrl/centralfacilities/poly
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• Møller, Søren Pape. CDS-Apparatus. 2006.
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• Computed circular dichroism spectra for the
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