Molecular Analysis of Changes in DNA Binding Affinity and Bending Extent Induced by the Mutations on the Aromatic Amino Residues in Cren7.

Proteins from Crenarchaeal organisms exhibit remarkable thermal stability. The aromatic amino acids in Cren7, a Crenarchaeal protein, regulate protein stability and further modulate DNA binding and its compaction. Specific aromatic amino acids were mutated, and using spectroscopic and theoretical approaches, we have examined the effect of the mutation on the structure, DNA binding affinity, and DNA bending ability of Cren7. The mutants were compared to the structure and function of wild-type (WT) Cren7. The reverse titration profiles were analysed by a noncooperative McGhee-von Hippel model to estimate affinity constant (K) and binding site size (n) associated with binding to the DNA. The biolayer interferometry (BLI) measurements showed that the binding affinity decreased at higher salt concentrations. For theoretical analysis of the extent of DNA bending, radius of gyration and bending angle were compared for WT and mutants. The time evolution of order parameters based on the translational and rotational motion of tryptophan residue (W26) was used for the qualitative detection of stacking interactions between W26 of Cren7 and DNA nucleobases. It was observed that the orientation of W26 in F41A favored the formation of a new lone pair-lone pair interaction between DNA and Cren7. Consequently, in thermostable proteins, the aromatic residues at the terminus maintain structural stability, whereas the residues at the core optimize the degree of DNA bending and compaction.