A novel molecular reporter for probing protein DNA recognition: An optical spectroscopic and molecular modeling study

[Display omitted] •Fluorescence lifetime of LV2 in H1 increases significantly upon interaction with DNA.•Rotational relaxation time of LV2 in H1 increases nearly four-fold upon interaction with DNA.•Binding of LV2 in the interface of H1-DNA complex is energetically most favourable relative to H1 or DNA. A novel benzo[a]phenoxazine-based fluorescent dye LV2 has been employed as a molecular reporter to probe recognition of a linker histone protein H1 by calf-thymus DNA (DNA). Fluorescence lifetime of LV2 buried in the globular domain of H1 (∼2.1 ns) or in the minor groove of DNA (∼0.93 ns) increases significantly to 2.65 ns upon interaction of the cationic protein with DNA indicating formation of the H1-DNA complex. The rotational relaxation time of the fluorophore buried in the globular domain of H1 increases significantly from 2.2 ns to 8.54 ns in the presence of DNA manifesting the recognition of H1 by DNA leading to formation of the H1-DNA complex. Molecular docking and molecular dynamics (MD) simulations have shown that binding of LV2 is energetically most favourable in the interface of the H1-DNA complex than in the globular domain of H1 or in the minor groove of DNA. As a consequence, orientational relaxation of the LV2 is significantly hindered in the protein-DNA interface compared to H1 or DNA giving rise to a much longer rotational relaxation time (8.54 ns) in the H1-DNA complex relative to that in pure H1 (2.2 ns) or DNA (5.7 ns). Thus, via a significant change of fluorescence lifetime and rotational relaxation time, the benzo[a]phenoxazine-based fluorescent dye buried within the globular domain of the cationic protein, or within the minor groove of DNA, reports on recognition of H1 by DNA.