Ultrafast differential flexibility of Cro-protein binding domains of two operator DNAs with different sequences

Abstract

The nature of the interface of specific protein-DNA complexes has attracted immense interest in contemporary molecular biology. Although extensive studies on the role of flexibility of DNA in the specific interaction in the genetic regulatory activity of lambda Cro (Cro-protein) have been performed, the exploration of quantitative features remains deficient. In this study, we have mutated (site directed mutagenesis: SDM) Cro-protein at the 37th position with a cysteine residue (G37C) retaining the functional integrity of the protein and labelled the cysteine residue, which is close to the interface, with a fluorescent probe (AEDANS), for the investigation of its interface with operator DNAs (OR3 and OR2). We have employed picosecond resolved polarization gated fluorescence spectroscopy and the well known strategy of solvation dynamics for the exploration of physical motions of the fluorescent probes and associated environments, respectively. Even though this particular probe on the protein (AEDANS) shows marginal changes in its structural flexibility upon interaction with the DNAs, a non-covalent DNA bound probe (DAPI), which binds to the minor groove, shows a major differential alteration in the dynamical flexibility in the OR3-Cro complex when compared to that of the OR2 complex with the Cro-protein. We attempt to correlate the observed significant structural fluctuation of the Cro-protein binding domain of OR3 for the specificity of the protein to the operator DNA.

Multispectroscopic and Theoretical Exploration of the Comparative Binding Aspects of Bioflavonoid Fisetin with Triple- and Double-Helical Forms of RNA

“…To obtain additional insight into the microenvironment around probe FTN, the time-resolved fluorescence anisotropy decay, , r ( t ), measurement of FTN in the absence and presence of the triplex- and duplex-bound environment has been performed. The time-resolved fluorescence anisotropy decay profiles of FTN in the bulk aqueous buffer solution and in the RNA-bound state are displayed in Figure , and the corresponding data are presented in Table .…”

Elucidating Electronic Structure Variations in Nucleic Acid-Protein Complexes Involved in Transcription Regulation Using a Tight-Binding Approach

“…Ultrafast electron transfer occurs during the recognition of various DNA sequences by a DNA-binding protein with distinct dynamic conformations. (39)(40)(41)(42)(43)(44) DNA damage and repair mechanisms involve electron transport. For instance, positive charge transfer can promote oxidative damage to guanine in DNA, which may be related to the presence of mutation sites in the genome.…”

Induced circularly polarized luminescence for revealing DNA binding with fluorescent dyes

“…TO is a well-known fluorescent stain of the cyanine family that intercalates between DNA base-pairs, [31] whereas DAPI shows more complex behaviour but mainly yields groove-bound DNA complexes [31][32][33] and is used as minor-groove binding probe. [34,35] By using our protocol for recording CPL data, [12,30] we will show also some limitations of this technique and discuss the main origin of the artefacts standing behind. In addition, induced ECD (ICD) as a complementary spectroscopy to CPL will be also investigated for monitoring DNA-dye binding process.…”