Author ORCID Identifier

https://orcid.org/0000-0003-4749-6878

Date of Award

17-8-2025

Document Type

Thesis

School

School of Chemical & Biotechnology

Programme

Ph.D.-Doctoral of Philosophy

First Advisor

Dr.Vigneshwar Ramakrishnan

Keywords

Protein-DNA Interactions, EcoRI Star Activity, Molecular Dynamics Simulations, Osmolytes Glycerol, DMSO DNA Conformations

Abstract

EcoRI is a type II restriction endonuclease that has been widely used as a model system to study protein-DNA interaction owing to its high degree of specificity. This enzyme does not recognize even a single basepair change in its recognition sequence, (GAATTC)2, under normal conditions. However, in the presence of osmolytes, such as glycerol and DMSO, the specificity of EcoRI is slightly relaxed and recognizes sequences that differ from its cognate sequence in the first basepair position. This relaxed specificity has been attributed to the dehydration of the EcoRI-DNA which presumably results in tighter complex formation and subsequent catalysis. However, it may be noted this is insufficient to explain the lack of recognition of DNA sequences that differ at second position or sequences that differ by more than one basepair.

In this thesis, we hypothesized that the relaxation in specificity is not only because of the dehydration of the EcoRI-DNA interface, but also because of its combined effect on the free EcoRI, free DNA and the EcoRI-DNA complex. To investigate this, we performed molecular dynamics simulations of free EcoRI, free DNA and the protein-DNA complex in the presence and absence of different concentrations of two osmolytes, viz., glycerol and DMSO. We used noncognate DNA sequences that differ from the cognate sequence in the first basepair (CAATTC, AAATTC and TAATTC) and as well used a non-specific sequence (TAGCTA) in our study. Our results show the following: (i) In free DNA, there is a sequence-dependent dehydration of the DNA sequence. This is also associated with a transition of the DNA conformation from the BI to BII state which may facilitate the binding of EcoRI.

Further, we observed that while glycerol interacts with DNA directly, DMSO does not as much as glycerol indicating that the two osmolytes may be exerting their action through different modes. Further analyses show that DMSO probably facilitates protein binding by the entropic advantage of the release of water, whereas Glycerol may facilitate protein binding through an entropically favourable displacement of glycerol, contributing to a net decrease in the free energy of binding (ΔG). (ii) The essential dynamics of the free EcoRI is altered in the presence of osmolytes to that similar to that when bound to cognate DNA.

Further, osmolytes slow down the tumbling motion of the interfacial waters, which, in turn, slows down the break-and-make of hydrogen bonds of the interfacial waters with functionally important residues. These results point to the idea that osmolytes may poise the EcoRI for binding to DNA sequences. (iii) In EcoRI-DNA complex systems, our results shows that the osmolyte-induced dehydration of the protein-DNA interface is associated with a number of attendant changes including retarded water dynamics, restored DNA kinking, partially restored protein-DNA hydrogen bonds and altered conformational landscape of EcoRI.

These attendant changes possibly help in relaxing the protein-DNA specificity. The gamut of changes in the structure and dynamics of the biomolecules in the presence of osmolytes not only shows the complex interplay underlying in the osmolyte-EcoRI-DNA-water systems, but also provides a framework to understand the extent of relaxation of protein-DNA specificity in the presence of osmolytes in general. In other words, this thesis contributes the literature on EcoRI-DNA specificity as well as points out to the need to dissect and understand the complex interplay that underlies biomolecular recognition in the presence of other cosolutes such as osmolytes.

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