Enkhbaatar, L. | Search bioRxiv

bioRxiv2026

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.

biophysics↗

bioRxiv2023

Using transition density models to interpret experimental optical spectra of exciton-coupled cyanine (iCy3)2 dimer probes of local DNA conformations at or near functional protein binding sites

Exciton-coupled chromophore dimers are an emerging class of optical probes for studies of site-specific biomolecular interactions. Applying accurate theoretical models for the electrostatic coupling of a molecular dimer probe is a key step for simulating its optical properties and analyzing spectroscopic data. In this work, we compare experimental absorbance and circular dichroism (CD) spectra of internally-labeled (iCy3)2 dimer probes inserted site-specifically into DNA fork constructs to theoretical calculations of the structure and geometry of these exciton-coupled dimers. We compare transition density models of varying levels of approximation to determine conformational parameters of the (iCy3)2 dimer-labeled DNA fork constructs. By applying an atomistically detailed transition charge (TQ) model, we can distinguish between dimer conformations in which the stacking and tilt angles between planar iCy3 monomers are varied. A major strength of this approach is that the local conformations of the (iCy3)2 dimer probes that we determined can be used to infer information about the structures of the DNA framework immediately surrounding the probes at various positions within the constructs, both deep in the duplex DNA sequences and at sites at or near the DNA fork junctions where protein complexes bind to discharge their biological functions.

biophysics↗