Gene 32 protein (gp32) from bacteriophage T4 is a sequence-nonspecific single-strand (ss) nucleic acid binding protein which binds highly cooperatively to ss nucleic acids. The N-terminal "B" or basic domain (residues 1-21) is known to be required for highly cooperative binding by gp32 (where K(app) = K(int) omega, omega > or = 500), since its removal results in a protein which binds ss nucleic acids noncooperatively (omega = 1). In this paper, we probe the molecular details of cooperative binding by gp32 by physicochemical characterization of a set of four single amino acid substitution mutants of Arg4: Lys4 (R4K gp32), Gln4 (R4Q gp32), Thr4 (R4T gp32), and Gly4 (R4G gp32). The qualitative ranking of binding affinities to poly(A) is wild-type > or = R4K > R4Q > R4T > R4G > gp32-B (gp32 lacking the first 21 amino acids). The occluded site size is n(app) = 7.5 +/- 0.5 for all gp32s. Resolution of K(int) and omega for wild-type, R4K, R4Q, and R4T gp32s was estimated under conditions of low lattice saturation (v < or = 0.011) using multiple reverse fluorescence titrations collected at 10 mM Tris-HCl, pH 8.1, 20 degrees C, and a NaCl concentration where K(app) was (2-4) x 10(6) M-1 for each gp32 on the ribohomopolymer poly(A). Binding parameters for all gp32s were obtained directly or compared by conservative extrapolation of the [NaCl] dependence of K(app) to 0.20 M NaCl, 20 degrees C, pH 8.1. The magnitude of omega was then assumed not to vary with [NaCl] (shown for R4T gp32), allowing estimation of K(int) at 0.20 M NaCl. We find that R4K gp32 binds to poly(A) with an overall affinity (K(app)) which is 2-3-fold lower than wild-type gp32, while omega for each molecule seems indistinguishable (wild-type gp32, omega approximately 800-1300; R4K gp32, omega approximately 600-1200). Surprisingly, R4Q gp32 is characterized by an omega also not readily distinguishable from the wild-type and R4K proteins (omega approximately 800-4400), while K(app) is reduced about 10-fold. This mutant also shows a significantly reduced [NaCl] dependence of the binding to poly(A). R4T gp32 binds about 10-fold weaker than the Q mutant. It exhibits an omega ranging from 300 to 700 and a substantially reduced [NaCl] dependence (delta log K(int)/delta log [NaCl] = -1.4 from 0.10 to 0.20 M NaCl), indicative of significant perturbations in both K(int) and omega terms.(ABSTRACT TRUNCATED AT 400 WORDS)