Binding of Cationic (+4) Alanine- and Glycine-Containing Oligopeptides to Double-Stranded DNA: Thermodynamic Analysis of Effects of Coulombic Interactions and R-Helix Induction ² S. Padmanabhan, ‡,§,| Wentao Zhang, ‡,§ Michael W. Capp, § Charles F. Anderson, § and M. Thomas Record, Jr.* ,§,⊥ Departments of Chemistry and Biochemistry, UniVersity of WisconsinsMadison, Madison, Wisconsin 53706 ReceiVed NoVember 27, 1996; ReVised Manuscript ReceiVed February 14, 1997 X ABSTRACT: Coulombic interactions and coupled conformational changes make important contributions to stability and specificity of many protein-nucleic acid complexes. As models of these phenomena in simpler systems, we have investigated the binding to mononucleosomal (160 base-pair) calf thymus DNA of a high charge density (compact) 5-residue (+4) oligopeptide (with 4 lysines and 1 tryptophan) and of four lower charge density (extended) 17-residue (+4) oligopeptides (each with 4 lysines, 10-12 alanines, 0-2 glycines, and 1 tryptophan). The fractional helicity (f h ) of each oligopeptide before and after DNA binding was determined using circular dichroism. At low univalent cation concentration ([M + ] ) 6.4 mM), binding to DNA increases f h significantly for all but one of the extended oligopeptides. Oligopeptide- DNA binding constants (K obs ) and apparent binding site sizes (n) were quantified using the noncooperative McGhee-von Hippel isotherm to fit tryptophan fluorescence quenching data. For each of the oligopeptides studied, n is found to be approximately equal to four, the number of lysine charges. In the range 6.4 mM e [M + ] e 21.5 mM, power dependences of K obs on [M + ](SK obs ≡ d log K obs /d log[M + ]) for all 17- residue (+4) oligopeptides are similar with an average value of -3.7 ( 0.4, which is indistinguishable (outside uncertainty) from the value obtained here for the compact (+4) oligopeptide and from values reported elsewhere for another compact tetralysine and for spermine (+4). Our results are consistent with the conclusion that the nonspecific binding to DNA of all these tetravalent ligands is driven primarily by coulombic interactions. At any [M + ] investigated, values of K obs for the four extended (+4) oligopeptides differ by less than an order of magnitude, but all are 1-2 orders of magnitude less than values of K obs for two compact (+4) oligopeptides and for spermine. The differences in K obs for oligopeptide-DNA complexes, which all have similar n and similar SK obs , indicate that when an extended oligopeptide binds to DNA it becomes more compact as a result of conformational changes, such as the additional R-helix formation detected by circular dichroism. Binding of proteins to DNA is driven in large part by the favorable thermodynamic consequences of reducing DNA polyanionic charge density (polyelectrolyte effect) and of burying nonpolar surface (hydrophobic effect) [see Record et al. (1991) and Spolar and Record (1994)]. Local protein folding transitions and other conformational changes, driven by binding free energy, create functionally important parts of the specific protein-DNA interface and give rise to a characteristic thermodynamic signature (Spolar & Record, 1994). In particular, R-helix formation is coupled to specific binding of many gene regulatory proteins and of oligopep- tides corresponding to the DNA-binding regions of various proteins (Arrowsmith et al., 1990; O’Neill et al., 1990, 1991; Talanian et al., 1990; Zhang et al., 1994; Lewis et al., 1996) and restriction enzymes (McClarin et al., 1986; Jen-Jacobson et al., 1986; Newman et al., 1995). For proteins that interact nonspecifically with DNA, smaller increases in helicity induced by binding have been reported (Arrowsmith et al., 1990; O’Neill et al., 1990, 1991; Zhang et al., 1994; Percipalle et al., 1995). Johnson et al. (1994) demonstrated that increases in R-helicity of low charge density (extended) lysine-containing alanine oligopeptides [as determined by circular dichroism (CD) 1 ] occur upon nonspecific binding to duplex DNA oligonucleotides. High charge density (compact) lysine oligopeptides (with net charge z ranging from 3 to 10 positive charges) have been used as model systems in investigations of salt concentration effects on the thermodynamics of protein-DNA interactions (Latt & Sober, 1967a,b; Lohman et al., 1980; Mascotti & Lohman, 1990, 1992, 1993; Zhang et al., 1996). For compact and extended DNA-binding oligopeptides, more systematic and quantitative comparisons should provide better understanding of how oligopeptide charge, structure, and helix-forming propensity affect the thermodynamic parameters that characterize their nonspecific binding to DNA. In the present study, we characterize and compare the nonspecific binding to double-helical 160 base-pair (bp) mononucleosomal calf thymus DNA (CT DNA) of five oligopeptides, specified in Table 1. These model (+4) ligands, one compact (5-residue) and four extended (17- ² This research was supported by NIH Grant GM 34351. * Corresponding author. ‡ The first two authors contributed equally to this study. § Department of Chemistry. | Present address: Instituto de Estructura de la Materia “Rocasolano”, CSIC, 119 Serrano, 28020 Madrid, Spain. ⊥ Department of Biochemistry. X Abstract published in AdVance ACS Abstracts, April 15, 1997. 1 Abbreviations: CD, circular dichroism; bp, base pair; CT DNA, mononucleosomal calf thymus DNA (160 bp); ds, double-stranded; [DNAP], DNA phosphate (monomer) concentration. 5193 Biochemistry 1997, 36, 5193-5206 S0006-2960(96)02927-3 CCC: $14.00 © 1997 American Chemical Society