Interactions%20of%20Charged%20Peptides%20with%20Polynucleic%20Acids

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Interactions ofCharged Peptides with Polynucleic
Acids
David P. Mascotti John Carroll University Departme
nt of Chemistry University Heights, OH 44118
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Original Purposes
1) Provide a Model System for the Salt Dependence
of Protein-Nucleic Acid Interactions 2)
Obtain a Thermodynamic Basis for Charged
Ligand-Nucleic Acid Interactions 3) Test Some
Polyelectrolyte Theories
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Protein-Nucleic Acid InteractionsA Cartoon
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General Effects of Salt on Charged Ligand-Nucleic
Acid Equilibria Linked Function Analysis
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Link to collapsed structures
? collapsed LD
L D ? LD
These studies
Future studies
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Predictions
Simple oligocations binding to polynucleotides,
in the absence of anion or water
rearrangement, Dc Z Y or Z (based on
counterion condensation hypothesis) What is Y?
Y 1 - (2x)-1 (where, x q2/ekTb) and
that means? Y the fraction of a cation
thermodynamically bound per phosphate to
relieve repulsion
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Oligopeptides
Lys-Trp-(Lys)p-NH2 (KWKp-NH2) Z p 2 (when
fully protonated) Lys-Trp-(Ile)2-(Lys)2-NH2
(KWI2K2-NH2) Z 4 (when fully
protonated) Lys-Trp-(Lys)p-CO2 (KWKp-CO2) Z
p 1 (when fully protonated) Arg-Trp-(Arg)p-NH2
(RWRp-NH2) Z p 2 (when fully
protonated) Arg-Trp-(Arg)p-CO2 (RWRp-CO2) Z
p 1 (when fully protonated)
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Fluorescence Quenchingof Tryptophan
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Light Scatter
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Calculation of Binding Isotherms from
Fluorescence Quenching Data
Qobs (Finit - Fobs)/ Finit Qobs/Qmax
Lb/Lt Qmax Qobs at Lb/Lt 1 n Lb/Dt
(Qobs/Qmax)(Lt/Dt) Lf Lt - n Dt
(Extent of Quenching is proportional to extent of
peptide binding)
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Treatment of overlapping binding sites for
nonspecific, noncooperative ligand-nucleic acid
interactions.
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Sample Reverse Titrations
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Saltback Titrations
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Sample of vant Hoff Analysis for Binding of
KWK4-NH2 to Poly(U) as a Function of salt
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Dependence of Kobs on Salt Concentration for
Oligolysine-poly(U) Interactions
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Salt Dependence of Kobs vs.Oligolysine Charge y
0.7 for poly(U)
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The Salt Dependence of Kobs for
Oligolysine-poly(U) Interactions is Due to
Cation Release
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The Salt Dependence of Kobs isIndependent of
Cation Type
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Changes in Free Energy, Enthalpy and Entropy as
Functions of Salt Concentration
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Correlation of Qmax to Standard State
Thermodynamic Quantities for Oligolysines Binding
to Poly(U)
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Qmax varies with PolynucleotideType and Peptide
Charge
poly(dT)
poly(U)
poly(A)
poly(C)
dsDNA
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Correlation of Qmax with DG?(1M) for
KWK4-NH2-polynucleotide Interactions
Qmax
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Salt Dependence of Kobs for OligolysinesBinding
to Different Homopolynucleotides
Y0.68
Y0.78
Y0.68
Y0.82
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Effect of Anion Type on the Dependence ofKobs on
Salt for RWR4-NH2 binding to poly(U)
NaF
NaCl
KOAc
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Dependence of Kobs on salt concentration for
Oligoarginine-poly(U) interactions Comparison to
Oligolysines
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And now, something thatwas supposed to be
simple...

• Effect of dielectric constant on y.
• Y 1 - (2x)-1 (where, x q2/ekTb)
• Therefore, Zy (i.e., slope of logKobs/logsalt)
• should increase with decreasing e.

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Salt dependence of E. coli SSB-poly(U)
Interactions with and without Glycerol
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Salt Dependence of Kobs as a Function of Solution
Dielectric
KWK2-NH2 binding to poly(U) pH6, 25C
-SKobs vs. e
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The Effect of the Cosolvent on Kobs
KWK2-NH2 binding to poly(U) at 29 mM KOAc, pH6,
25C
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KWK2-NH2- and KWI2K2-NH2-poly(U) Interactions
Various Cosolvents
KWK2-NH2 -poly(U) at 25oC
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Thermodynamic Data and Salt Dependence for Each
Cosolvent
All thermodynamic data was collected at 40.1 mM
M and all saltbacks were performed at 25oC.
?Ho values are in kcal/mol and ?So values are in
cal/mol. Estimated error in ?Ho is ?1.5
kcal/mol and in ?So is ?5 cal/mol Note it was
found that DH was independent of salt
concentration
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Interpreting the Ethanol data for KWK2-NH2 and
KWI2K2-NH2

• Ethanol induces a steeper -SKobs for KWK2-NH2.
  Stronger anion binding? If so, hydration of the
  anion upon release ? more favorable ?H.
• or more favorable ?H in ethanol could be
  explained by ethanol promoting water molecules
  being released from the peptide or RNA.2
• d ln(Kobs)/ dosmolal -?nw/55.6
• from this equation, estimate that approximately
  12 water molecules are released from the
  peptide-RNA complex
• Upon being released these water molecules may
  form stronger hydrogen bonds with other water
  molecules than with the RNA or peptide
• There is also a more favorable ?H when ethanol is
  used as the cosolvent with KWI2K2-NH2. However,
  the SKobs is not as affected. Why?
  (incongruent with first argument above, better
  for second)

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Future Studies

• More highly charged peptides (e.g., KWK29-NH2)
• Arginine-based peptides
• Determine anion effect with MeOH EtOH
• New ITC and DSC Calorimeters
• -could be used to help determine collapse
  step
• Other osmolytes?
• Volume exclusion agents?

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Take-Home Messages

• Charged Peptide-nucleotide interactions useful
  data set
• for comparison to protein-DNA and -RNA
  interactions.
• Inclusion of hydrophobic residues in the peptides
  can
• affect -SKobs
• The nature of the anion may not be trivial for
  highly
• charged peptides, especially in hydrophobic
  environments
• Slopes of logKobs vs. logsalt plots must be
  dissected
• to interpret Z correctly.

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Acknowledgements

• John Carroll University
• National Science Foundation
• Huntington and Codrington Foundations
• Dreyfus Foundation Special Awards in Chemistry
• James Bellar, Niki Kovacs, Amy Salwan, Michael
  Iannetti

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Stop!
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Effect of the Number of Tryptophanson Ion
Displacement
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Dependence of Thermodynamic Properties on Number
of Tryptophans
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Standard State Thermodynamic Quantitiesof
Oligolysines Binding to ssRNA and
ssDNADependence on Peptide Charge
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Cuvette Adhesion