Publication: Spectroscopic Understanding Of Structure, Function and Dynamics in Biomolecules ; From Ensemble to single Molecule
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2024-10-31
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Indian Institute of Technology, Jodhpur
Abstract
Biomolecules are intricate entities in which the structure, function, and dynamics are
intrinsically interrelated. The function of a biomolecule not only depends on the structure but also the dynamics in real-time. Deciphering the interlink among structure, function and dynamics is critical for elucidating the molecular mechanisms that drive important biological processes at the cellular level. Single molecule fluorescence spectroscopy is ideal for understanding these dynamics because it enables observation of structural changes, molecular interactions, and conformational transitions of a single specific molecule in real-time. In order to gain a comprehensive understanding of the interactions and functions of biomolecules in biological systems, our research employs a range of spectroscopic techniques to investigate these dimensions. In the first part ofmy thesis, we investigated the interactions between proteins and hydrophobic drugs in presence of drug carrier.We used fluorescence spectroscopy and molecular dynamics simulations to show how diflunisal interacts with cyclodextrin complexes in the presence of protein. The observations enhance the development of more effective pharmaceutical delivery methods. We studied how curcumin interacts with bovine serum albumin (BSA) in presence of micelle carrier and found that strong hydrophobic interactions drive the binding,whichmay help us understand how well curcumin works in clinical trials. In this thesis, we also explore the single-molecule technique to understand molecular heterogeneity and complex dynamics, which ensemble studies typically obscure. In order to observe conformational changes in molecules in real-time with high spatial-temporal resolution at the single-molecule level, we built a prism-based total internal reflection microscope setup to perform single molecule fluorescence resonance energy transfer (smFRET) experiments. We use the smFRET technique to study the conformational heterogeneity in a DNA Holliday Junction (HJ), with a thorough statistical analysis we have found patterns of resonance, coherence, and decoherence like intermittent conformational dynamics. In the subsequent chapter we also performed smFRET experiments and molecular simulations to investigate the kinetic choreography and preferential stability of HJ conformations under ionic conditions that closely mimic the physiological environment relevant to cellular biology. Our findings predict the prevalence of three distinct conformational macrostates in HJ dynamics. Using a kinetic network model based on a multi-order time correlation function (TCF), we delineate the thermodynamic parameters that govern the heterogeneous dynamics as a function of divalent ion concentration. Stabilization of conformations due to the ionic environment and activation barriers concertedly affect the transition rates between open and closed conformations. Furthermore, we observed a significant enhancement of Mg2+ condensation in the central region of the HJ rather than the branch ends, leading toward a plausible conclusion that the observed differential stability of conformational statesmay be governed by the junction region of the HJ rather than the duplex branches. This study provides new insights into the complex interplay between the ionic environment and HJ dynamics, offering a comprehensive understanding of HJ behavior under conditions relevant to cellular biology, and its roles in key biological process create the heterogeneous nature of life....
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Agarwala, Pratibha (2020).Spectroscopic Understanding Of Structure, Function and Dynamics in Biomolecules ; From Ensemble to single Molecule(Doctor's thesis).Indian Institute of Technology, Jodhpur