The N-terminal B-domain of T4 gene 32 protein modulates the lifetime of cooperatively bound Gp32-ss nucleic acid complexes.
The N-terminal basic or B-domain (residues 1-21) of bacteriophage T4 gene 32 protein (gp32) provides a major determinant for highly cooperative binding by gp32 to single-stranded (ss) nucleic acids at equilibrium. In order to gain mechanistic insight into N-terminal domain function, the kinetics of dissociation of wild-type and previously characterized B-domain substitution mutant gp32s (R4K, R4Q, and K3A) from the model ribohomopolymer, poly(A), have been investigated under solution conditions identical to those used for equilibrium studies [Villemain, J. L., & Giedroc, D. P. (1993) Biochemistry 32, 11235-11246; Villemain, J. L., & Giedroc, D. P. (1996) J. Biol. Chem. 271, 27623-27629]. The dissociation of cooperatively bound gp32-poly(A) complexes was induced by sodium chloride concentration jumps and monitored by an increase in tryptophan fluorescence upon dissociation of the protein from poly(A) using stopped-flow techniques. The apparent dissociation rate constant, kd(app), for all mutant proteins studied was found to depend strongly on the initial fractional saturation of poly(A) just as was found previously for wild-type gp32. This permitted application of Lohman's model for the irreversible dissociation of cooperatively bound gp32-nucleic acid complexes [Lohman, T. M. (1983) Biopolymers 22, 1697-1713] from which the molecular rate constant, ke, the rate of dissociation of a protein monomer from teh end of a gp32-ss nucleic acid complex or protein cluster, could be determined. From the [NaCl]-dependence of kd(app), ke determined at 0.45 M NaCl, pH 8.1, 20 degrees C, was found to be 62 +/- 23, 78 +/- 8, 328 +/- 36, and 384 +/- 34 s-1 for wild-type, R4K, K3A, and R4Q gp32s, respectively. With the exception of R4K gp32, we find a striking correlation between the relative magnitudes of ke and Kapp, suggesting that the molecular defect in the equilibrium binding properties of the N-terminal domain mutants resides in the increased rate at which gp32 monomers dissociate from singly contiguous binding sites at the ends of clusters. The bimolecular association rate constant measured for wild-type gp32 and a weakly binding B-domain mutant, R4T gp32, to poly(dT) was found to be nearly identical, further evidence that the primary defect is in the dissociation reaction. We conclude that the N-terminal domain strongly modulates the lifetime of cooperatively bound gp32-polynucleotide complexes. The mechanistic and functional implications of these findings are discussed.
Department of Biochemistry and Biophysics, Texas A&M University, College Station 77843-2128, USA.
J L
JL
Villemain
D P
DP
Giedroc
D001120:Arginine / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry*; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D012965:Sodium Chloride / Q000737:chemistry; D013329:Structure-Activity Relationship; D014764:Viral Proteins / Q000737:chemistry*
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D014764:Viral Proteins; C060035:gp32 protein, Enterobacteria phage T4; D012965:Sodium Chloride; D001120:Arginine; D008239:Lysine