title: Physical and structural basis for the strong interactions of the -ImPy- central pairing motif in the polyamide f-ImPyIm.
abstract: The polyamide f-ImPyIm has a higher affinity for its cognate DNA than either the parent analogue, distamycin A (10-fold), or the structural isomer, f-PyImIm (250-fold), has for its respective cognate DNA sequence. These findings have led to the formulation of a two-letter polyamide "language" in which the -ImPy- central pairings associate more strongly with Watson-Crick DNA than -PyPy-, -PyIm-, and -ImIm-. Herein, we further characterize f-ImPyIm and f-PyImIm, and we report thermodynamic and structural differences between -ImPy- (f-ImPyIm) and -PyIm- (f-PyImIm) central pairings. DNase I footprinting studies confirmed that f-ImPyIm is a stronger binder than distamycin A and f-PyImIm and that f-ImPyIm preferentially binds CGCG over multiple competing sequences. The difference in the binding of f-ImPyIm and f-PyImIm to their cognate sequences was supported by the Na(+)-dependent nature of DNA melting studies, in which significantly higher Na(+) concentrations were needed to match the ability of f-ImPyIm to stabilize CGCG with that of f-PyImIm stabilizing CCGG. The selectivity of f-ImPyIm beyond the four-base CGCG recognition site was tested by circular dichroism and isothermal titration microcalorimetry, which shows that f-ImPyIm has marginal selectivity for (A.T)CGCG(A.T) over (G.C)CGCG(G.C). In addition, changes adjacent to this 6 bp binding site do not affect f-ImPyIm affinity. Calorimetric studies revealed that binding of f-ImPyIm, f-PyImIm, and distamycin A to their respective hairpin cognate sequences is exothermic; however, changes in enthalpy, entropy, and heat capacity (DeltaC(p)) contribute differently to formation of the 2:1 complexes for each triamide. Experimental and theoretical determinations of DeltaC(p) for binding of f-ImPyIm to CGCG were in good agreement (-142 and -177 cal mol(-)(1) K(-)(1), respectively). (1)H NMR of f-ImPyIm and f-PyImIm complexed with their respective cognate DNAs confirmed positively cooperative formation of distinct 2:1 complexes. The NMR results also showed that these triamides bind in the DNA minor groove and that the oligonucleotide retains the B-form conformation. Using minimal distance restraints from the NMR experiments, molecular modeling and dynamics were used to illustrate the structural complementarity between f-ImPyIm and CGCG. Collectively, the NMR and ITC experiments show that formation of the 2:1 f-ImPyIm-CGCG complex achieves a structure more ordered and more thermodynamically favored than the structure of the 2:1 f-PyImIm-CCGG complex.
authors.affiliation: Department of Chemistry, Furman University, Greenville, South Carolina 29613, USA.
authors.forename: Karen L
authors.initials: KL
authors.lastname: Buchmueller
authors.forename: Suzanna L
authors.initials: SL
authors.lastname: Bailey
authors.forename: David A
authors.initials: DA
authors.lastname: Matthews
authors.forename: Zarmeen T
authors.initials: ZT
authors.lastname: Taherbhai
authors.forename: Janna K
authors.initials: JK
authors.lastname: Register
authors.forename: Zachary S
authors.initials: ZS
authors.lastname: Davis
authors.forename: Chrystal D
authors.initials: CD
authors.lastname: Bruce
authors.forename: Caroline
authors.initials: C
authors.lastname: O'Hare
authors.forename: John A
authors.initials: JA
authors.lastname: Hartley
authors.forename: Moses
authors.initials: M
authors.lastname: Lee
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D002151:Calorimetry; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry*; D018983:DNA Footprinting; D003850:Deoxyribonuclease I / Q000378:metabolism; D004214:Distamycins / Q000737:chemistry*; D007093:Imidazoles / Q000737:chemistry*; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D009691:Nucleic Acid Denaturation; D009757:Nylons / Q000737:chemistry*; D011758:Pyrroles / Q000737:chemistry*; D013816:Thermodynamics
chemical_list: D004214:Distamycins; D007093:Imidazoles; C516205:N-(2-(diethylamino)ethyl)-1-methyl-4-(1-methyl-4-(4-formamido-1-methylimidazole-2-carboxamido)pyrrole-2-carboxamido)imidazole-2-carboxamide; C487888:N-(2-(dimethylamino)ethyl)-1-methyl-4-(1-methyl-4-(4-formamido-1-methylpyrrole-2-carboxamido)imidazole-2-carboxamido)imidazole-2-carboxamide; D009757:Nylons; D011758:Pyrroles; C030000:stallimycin; D004247:DNA; D003850:Deoxyribonuclease I