Interactions of Charged Peptides with Polynucleic Acids

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

2. 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

3. Counterion Condensation Protein-Nucleic Acid Interactions:A Cartoon

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

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

6. Predictions 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

7. 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)

8. Fluorescence Quenchingof Tryptophan

9. Light Scatter

10. 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)

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

12. Sample “Reverse” Titrations

13. “Saltback” Titrations

14. Sample of van’t Hoff Analysis for Binding of KWK4-NH2 to Poly(U) as a Function of [salt]

15. Dependence of Kobs on Salt Concentration for Oligolysine-poly(U) Interactions

16. Salt Dependence of Kobs vs.Oligolysine Charge: y = 0.7 for poly(U)

17. The Salt Dependence of Kobs for Oligolysine-poly(U) Interactions is Due to Cation Release

18. The Salt Dependence of Kobs isIndependent of Cation Type

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

20. Correlation of Qmax to Standard State Thermodynamic Quantities for Oligolysines Binding to Poly(U)

21. Qmax varies with PolynucleotideType and Peptide Charge poly(dT) poly(U) poly(A) poly(C) dsDNA

22. Correlation of Qmax with DG(1M) for KWK4-NH2-polynucleotide Interactions Qmax

23. Salt Dependence of Kobs for OligolysinesBinding to Different Homopolynucleotides Y=0.78 Y=0.68 Y=0.68 Y=0.82

24. Effect of Anion Type on the Dependence ofKobs on [Salt] for RWR4-NH2 binding to poly(U) NaF NaCl KOAc

25. Dependence of Kobs on salt concentration for Oligoarginine-poly(U) interactions: Comparison to Oligolysines

27. 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/log[salt]) should increase with decreasing e.

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

29. Salt Dependence of Kobs as a Function of Solution Dielectric KWK2-NH2 binding to poly(U) pH6, 25°C -SKobs vs. e

30. The Effect of the Cosolvent on Kobs KWK2-NH2 binding to poly(U) at 29 mM KOAc, pH6, 25°C

31. KWI2K2-NH2 -poly(U) at 25oC KWK2-NH2- and KWI2K2-NH2-poly(U) Interactions: Various Cosolvents KWK2-NH2 -poly(U) at 25oC

32. 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

33. 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)/ 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)

34. 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?

35. 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. log[salt] plots must be dissected to interpret Z correctly.

36. 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

37. Stop!

38. Effect of the Number of Tryptophanson Ion Displacement

40. Dependence of Thermodynamic Properties on Number of Tryptophans

41. Standard State Thermodynamic Quantitiesof Oligolysines Binding to ssRNA and ssDNADependence on Peptide Charge

42. Cuvette Adhesion