Presentation on theme: "Interactions of Charged Peptides with Polynucleic Acids David P. Mascotti John Carroll University Department of Chemistry University Heights, OH 44118."— Presentation transcript:
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)
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.
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