The intrinsically disordered C-terminal domain of bacteriophage T4 gp32 amplifies pre-existing conformational heterogeneity at DNA replication junctions.

Selective molecular recognition of dynamic nucleic acid structures is fundamental to DNA replication, yet the physical mechanisms by which intrinsically disordered protein domains achieve this selectivity remain poorly understood. Here, we investigate how the intrinsically disordered C-terminal domain (CTD) of the bacteriophage T4 single-stranded DNA-binding protein gp32 contributes to the recognition of ss-dsDNA replication fork junctions. Using absorbance, circular dichroism, and two-dimensional fluorescence spectroscopy (2DFS) of model DNA replication junctions containing an exciton-coupled (Cy3) 2 probe, we quantified their average solution structures and conformational heterogeneity. Protein-free DNA junctions exhibit distinct conformational distributions that depend on ssDNA arm lengths and local base sequence, demonstrating that this heterogeneity exists prior to protein binding. Although both wild-type gp32 and the CTD-truncated mutant gp32 I* substantially remodel the average junction structure, only wild-type gp32 preserves and amplifies these differences in conformational heterogeneity. These results support a model in which the intrinsically disordered CTD promotes selective molecular recognition by redistributing populations among pre-existing DNA conformational states rather than by stabilizing a single protein-bound conformation. More broadly, our findings suggest that intrinsically disordered protein domains can recognize dynamic nucleic acid substrates by amplifying conformational heterogeneity already present within their intrinsic conformational landscapes.