Interactions of charged peptides with polynucleic acids
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Interactions of Charged Peptides with Polynucleic Acids. David P. Mascotti John Carroll University Department of Chemistry University Heights, OH 44118. Original Purposes. 1) Provide a Model System for the Salt Dependence of Protein-Nucleic Acid Interactions

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Interactions of charged peptides with polynucleic acids
Interactions ofCharged Peptides with Polynucleic Acids

David P. Mascotti

John Carroll University

Department of Chemistry

University Heights, OH 44118

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

Protein nucleic acid interactions a cartoon

Counterion Condensation

Protein-Nucleic Acid Interactions:A Cartoon

General Effects of Salt on Charged Ligand-Nucleic Acid Equilibria: Linked Function Analysis

Link to collapsed structures Equilibria: Linked Function Analysis

 collapsed LD

L + D  LD

These studies

Future studies

Predictions Equilibria: Linked Function Analysis

Simple oligocations binding to polynucleotides, in

the absence of anion or water rearrangement,

Dc = Z Yor 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

Oligopeptides Equilibria: Linked Function Analysis

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)

Fluorescence quenching of tryptophan
Fluorescence Quenching Equilibria: Linked Function Analysisof Tryptophan

Light Scatter Equilibria: Linked Function Analysis

Calculation of binding isotherms from fluorescence quenching data
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)

Treatment of Data

overlapping binding sites

for nonspecific,


ligand-nucleic acid


Sample of van’t Hoff Analysis for Binding Data

of KWK4-NH2 to Poly(U) as a Function of [salt]

Dependence of k obs on salt concentration for oligolysine poly u interactions
Dependence of K Dataobs on Salt Concentration for Oligolysine-poly(U) Interactions

Salt dependence of k obs vs oligolysine charge y 0 7 for poly u
Salt Dependence of K Dataobs vs.Oligolysine Charge: y = 0.7 for poly(U)

The Salt Dependence of K Dataobs for Oligolysine-poly(U) Interactions is Due to Cation Release

The salt dependence of k obs is independent of cation type
The Salt Dependence of K Dataobs isIndependent of Cation Type

Correlation of Q Salt Concentrationmax to Standard State Thermodynamic Quantities for Oligolysines Binding to Poly(U)

Q max varies with polynucleotide type and peptide charge
Q Salt Concentrationmax varies with PolynucleotideType and Peptide Charge






Correlation of q max with d g 1m for kwk 4 nh 2 polynucleotide interactions
Correlation of Q Salt Concentrationmax with DG(1M) for KWK4-NH2-polynucleotide Interactions


Salt dependence of k obs for oligolysines binding to different homopolynucleotides
Salt Dependence of K Salt Concentrationobs for OligolysinesBinding to Different Homopolynucleotides





Effect of anion type on the dependence of k obs on salt for rwr 4 nh 2 binding to poly u
Effect of Anion Type on the Dependence of Salt ConcentrationKobs on [Salt] for RWR4-NH2 binding to poly(U)




Dependence of K Salt Concentrationobs on salt concentration for Oligoarginine-poly(U) interactions: Comparison to Oligolysines

And now something that was supposed to be simple
And now, something that Salt Concentrationwas 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/log[salt])

    should increase with decreasing e.

Salt dependence of k obs as a function of solution dielectric
Salt Dependence of K without Glycerolobs as a Function of Solution Dielectric

KWK2-NH2 binding to poly(U)

pH6, 25°C

-SKobs vs. e

The Effect of the Cosolvent on K without Glycerolobs

KWK2-NH2 binding to poly(U)

at 29 mM KOAc, pH6, 25°C

Kwk 2 nh 2 and kwi 2 k 2 nh 2 poly u interactions various cosolvents

KWI without Glycerol2K2-NH2 -poly(U) at 25oC

KWK2-NH2- and KWI2K2-NH2-poly(U) Interactions: Various Cosolvents

KWK2-NH2 -poly(U) at 25oC

Thermodynamic data and salt dependence for each cosolvent
Thermodynamic Data and Salt Dependence for Each Cosolvent* without Glycerol

* 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

Interpreting the ethanol data for kwk 2 nh 2 and kwi 2 k 2 nh 2
Interpreting the Ethanol data for KWK without Glycerol2-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)/ d[osmolal] = -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)

Future studies
Future Studies without Glycerol

  • 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?

Take home messages
Take-Home Messages without Glycerol

  • 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. log[salt] plots must be dissected

    to interpret Z correctly.

Acknowledgements without Glycerol

  • John Carroll University

  • National Science Foundation

  • Huntington and Codrington Foundations

  • Dreyfus Foundation Special Awards in Chemistry

  • James Bellar, Niki Kovacs, Amy Salwan, Michael Iannetti

Stop! without Glycerol

Effect of the number of tryptophans on ion displacement
Effect of the Number of Tryptophans without Glycerolon Ion Displacement

Standard State Thermodynamic Quantities Tryptophansof Oligolysines Binding to ssRNA and ssDNADependence on Peptide Charge

Cuvette Adhesion Tryptophans