Characterization of a cooperativity domain mutant Lys3 → Ala (K3A) T4 gene 32 protein

Abstract

The N-terminal 'B' domain of T4 gene 32 protein contains a Lys3-Arg4- Lys5 sequence that has been postulated to provide a major determinant for cooperative binding. In this report, the equilibrium binding properties of a Lys3 → Ala substitution mutant of gp32 (K3A gp32) and described and compared to a set of substitution mutants of Arg4 previously described (Villemain, J. L., and Giedroc, D. P. (1993) Biochemistry 32, 11235-11246) and further characterized here. K3A gp32 exhibits binding behavior which mirrors that of R4Q gp32. Despite an 6-8-fold decrease in overall binding affinity (K(app) = K(int) x ω) at pH 8.1, 0.20 M NaCl, 20 °C, the magnitude of the cooperativity parameter is at most 2-3-fold smaller than that of the wild-type protein. The magnitude of ω is independent of salt concentration and type over the range in [NaCl] from 0.125 to 0.225 M and [NaF] between 0.20 and 0.32 M (log ω = 2.86 ± 0.19). For comparison, log ω for wild- type gp32 is 2.91 (± 0.21) resolved at 0.275 M NaCl and 3.39 ± 0.11 in [NaF] between 0.40 and 0.45 M. In contrast to ω, the [NaCl] dependence of K(app) is large and markedly nonlinear for both wild-type and K3A gp32s over a [NaCl] range extending from 0.05 M to 0.40 M NaCl. Modeling of the complete salt dependence of K(app) for wild-type, K3A, and R4T gp32s in NaCl and NaF with a simple ion-exchange model suggests that substitutions within the basic Lys3-Arg4-Lys5 sequence do not strongly modulate the net displacement of cations and anions upon poly(A) complex formation by gp32.