Interactions of Charged Peptides with Polynucleic Acids David P. Mascotti John Carroll University Department of Chemistry University Heights, OH 44118

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 Original Purposes

Protein-Nucleic Acid Interactions: A Cartoon Counterion Condensation

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

Link to collapsed structures L + D  LD These studies Future studies  collapsed LD

Predictions Simple oligocations binding to polynucleotides, in the absence of anion or water rearrangement,  c = Z   or Z (based on counterion condensation hypothesis) What is  ?  = 1 - (2  ) -1 (where,  = q 2 /  kTb) …and that means?  = the fraction of a cation “thermodynamically” bound per phosphate to relieve repulsion

Oligopeptides Lys-Trp-(Lys) p -NH 2 (KWK p -NH 2 ) Z = p + 2 (when fully protonated) Lys-Trp-(Ile) 2 -(Lys) 2 -NH2 (KWI 2 K 2 -NH 2 ) Z = 4 (when fully protonated) Lys-Trp-(Lys) p -CO 2 (KWK p -CO 2 ) Z = p + 1 (when fully protonated) Arg-Trp-(Arg) p -NH 2 (RWR p -NH 2 ) Z = p + 2 (when fully protonated) Arg-Trp-(Arg) p -CO 2 (RWR p -CO 2 ) Z = p + 1 (when fully protonated)

Fluorescence Quenching of Tryptophan

Light Scatter

Calculation of Binding Isotherms from Fluorescence Quenching Data Q obs = (F init - F obs )/ F init Q obs /Q max = L b /L t Q max = Q obs at L b /L t = 1 = L b /D t = (Q obs /Q max )(L t /D t ) L f = L t - D t (Extent of Quenching is proportional to extent of peptide binding)

Treatment of overlapping binding sites for nonspecific, noncooperative ligand-nucleic acid interactions.

Sample “Reverse” Titrations

“Saltback” Titrations

Sample of van’t Hoff Analysis for Binding of KWK 4 -NH 2 to Poly(U) as a Function of [salt]

Dependence of K obs on Salt Concentration for Oligolysine-poly(U) Interactions

Salt Dependence of K obs vs. Oligolysine Charge:  = 0.7 for poly(U)

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

The Salt Dependence of K obs is Independent of Cation Type

Changes in Free Energy, Enthalpy and Entropy as Functions of Salt Concentration

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

Q max varies with Polynucleotide Type and Peptide Charge dsDNA poly(C) poly(A) poly(U) poly(dT)

Correlation of Q max with  G  (1M) for KWK 4 -NH 2 -polynucleotide Interactions Q max

Salt Dependence of K obs for Oligolysines Binding to Different Homopolynucleotides  =0.68  =0.78  =0.68  =0.82

Effect of Anion Type on the Dependence of K obs on [Salt] for RWR 4 -NH 2 binding to poly(U) NaF KOAc NaCl

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

And now, something that was supposed to be simple... Effect of dielectric constant on .  = 1 - (2  ) -1 (where,  = q 2 /  kTb) Therefore, Z  (i.e., slope of logK obs /log[salt]) should increase with decreasing .

Salt dependence of E. coli SSB-poly(U) Interactions with and without Glycerol

Salt Dependence of K obs as a Function of Solution Dielectric -SK obs vs.  KWK 2 -NH 2 binding to poly(U) pH6, 25°C

The Effect of the Cosolvent on K obs KWK 2 -NH 2 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 KWK 2 -NH 2 -poly(U) at 25 o C KWI 2 K 2 -NH 2 -poly(U) at 25 o C

Thermodynamic Data and Salt Dependence for Each Cosolvent* * All thermodynamic data was collected at 40.1 mM [M + ] and all saltbacks were performed at 25 o C.  H o values are in kcal/mol and  S o values are in cal/mol. Estimated error in  H o is  1.5 kcal/mol and in  S o is  5 cal/mol Note: it was found that  H° was independent of salt concentration

Ethanol induces a steeper -SK obs for KWK 2 -NH 2. 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(K obs )/ d[osmolal] = -  n w /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 KWI 2 K 2 -NH 2. However, the –SK obs is not as affected. Why? (incongruent with first argument above, better for second) Interpreting the Ethanol data for KWK 2 -NH 2 and KWI 2 K 2 -NH 2

Future Studies More highly charged peptides (e.g., KWK 29 -NH 2 ) 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 Charged Peptide-nucleotide interactions: useful data set for comparison to protein-DNA and -RNA interactions. Inclusion of hydrophobic residues in the peptides can affect -SK obs The nature of the anion may not be trivial for highly charged peptides, especially in hydrophobic environments Slopes of logK obs vs. log[salt] plots must be dissected to interpret Z correctly.

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

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Effect of the Number of Tryptophans on Ion Displacement

Dependence of Thermodynamic Properties on Number of Tryptophans

Standard State Thermodynamic Quantities of Oligolysines Binding to ssRNA and ssDNA Dependence on Peptide Charge

Cuvette Adhesion