Computational Molecular Biology 2024, Vol.14, No.5, 182-190 http://bioscipublisher.com/index.php/cmb 183 years. We will explore various biophysical techniques for studying these interactions, emphasizing their contribution to our understanding of cellular processes and disease mechanisms. In addition, the impact of these interactions on drug discovery and the development of new therapeutic strategies will be discussed, aiming to provide a coherent understanding of the dynamic and complex nature of molecular interactions in biological systems and their technological applications. 2 Biophysical Techniques for Studying Molecular Interactions 2.1 X-ray crystallography X-ray crystallography is a cornerstone technique in structural biology, providing atomic-resolution structures of biomolecules. This method involves the crystallization of the molecule of interest and subsequent diffraction of X-rays through the crystal lattice. The resulting diffraction pattern is analyzed to determine the electron density and thus the three-dimensional structure of the molecule. X-ray crystallography has been instrumental in elucidating the structures of numerous proteins and nucleic acids, linking structural information to biological function and dynamics. 2.2 Nuclear magnetic resonance (NMR) spectroscopy NMR spectroscopy is another pivotal technique in structural biology, offering insights into the structure, dynamics, and interactions of biomolecules in solution. Unlike X-ray crystallography, NMR does not require crystallization, making it suitable for studying molecules in their native state. Recent advancements in solid-state NMR (ssNMR) have expanded its applications to include samples with static and dynamic disorder, such as lipid bilayers and protein aggregates. ssNMR provides complementary data to other structural techniques, enhancing our understanding of complex biological assemblies (Wel, 2018; Tsegaye et al., 2021). 2.3 Cryo-electron microscopy (Cryo-EM) Cryo-EM has emerged as a powerful tool for studying large macromolecular complexes that are challenging to analyze using X-ray crystallography or NMR. This technique involves flash-freezing samples in vitreous ice and imaging them using an electron microscope. Recent technological advancements have significantly improved the resolution of cryo-EM, allowing for near-atomic resolution structures. Cryo-EM is particularly valuable for studying dynamic and heterogeneous systems, providing detailed insights into the structural basis of biological mechanisms (Lerner et al., 2018; Tan and Carragher, 2020). 2.4 Surface plasmon resonance (SPR) SPR is an optical technique used to study ligand-analyte interactions in real-time without the need for labeling. It measures changes in the refractive index near a metal surface, which occur upon binding of molecules. SPR is widely used to investigate biomolecular interactions, including protein-protein, protein-DNA, and protein-membrane interactions. Recent advancements in SPR technology, such as multiplexed and regenerable biosensors, have enhanced its sensitivity and specificity, making it a valuable tool in both basic research and applied fields like drug discovery and GMO detection (Renaud et al., 2016). 2.5 Isothermal titration calorimetry (ITC) ITC is a thermodynamic technique that measures the heat change associated with molecular interactions, providing direct insights into binding affinities, stoichiometry, and thermodynamic parameters. It is a label-free method that can be used to study a wide range of interactions, including protein-ligand, protein-protein, and protein-DNA interactions. ITC is particularly useful in drug discovery for characterizing the binding properties of potential therapeutic compounds (Gavriilidou et al., 2022). 3 Applications of Biophysical Methods in Biological Research 3.1 Protein-protein interactions Protein-protein interactions (PPIs) are fundamental to numerous cellular processes, including signal transduction, cellular assembly, and enzymatic catalysis. Various biophysical methods have been developed to characterize these interactions, each with its strengths and limitations. Techniques such as mass spectrometry, nuclear magnetic resonance (NMR), and X-ray crystallography have been instrumental in mapping the interaction networks and
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