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Steady-State Fluorescence Spectroscopy as a Tool to Monitor Protein/Ligand Interactions

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Optical Spectroscopic and Microscopic Techniques

Abstract

Fluorescence spectroscopy is an ideal and powerful methodology for the potent and reliable study of protein–ligand interactions. Enhanced susceptibility accompanied with comparative easiness forms the prominent key factor for the application of fluorescence techniques in these studies. In fluorescence technique, protein–ligand interactions are often studied at very low concentrations compared to other optical methods with a thousandfold higher sensitivity. In this method, we measure the variation in quantum yield upon ligand binding, by observing variations in ligand fluorescence, intrinsic protein fluorescence, or fluorescence of covalently or noncovalently bound fluorescent probes that are sensitive to ligand binding. Sensitivity of a fluorescent ligand to the environment, energy transfer from protein to ligand producing reduction in protein fluorescence or amplification of ligand fluorescence, a conformational change in protein/ ligand on binding to the protein, etc., contribute to the changes in quantum yield upon ligand binding [1–3]. This chapter will outline a sketch of steady-state fluorescence techniques for defining molecular interactions and calculation of binding constants.

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Authors and Affiliations

  1. Department of Chemistry, School of Advanced Sciences, VIT, Vellore, Tamilnadu, India

    Roopa Kenoth & Ravi Kanth Kamlekar

  2. Division of Chemistry, School of Advanced Sciences, VIT, Chennai, Tamilnadu, India

    Balamurali M. M.

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  1. Roopa Kenoth
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  2. Balamurali M. M.
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  3. Ravi Kanth Kamlekar
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Correspondence to Ravi Kanth Kamlekar .

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Editors and Affiliations

  1. Biophysical and Protein Chemistry Laboratory, Department of Chemistry, National Institute of Technology (NIT) Rourkela, Rourkela, Odisha, India

    Harekrushna Sahoo

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Kenoth, R., M., B.M., Kamlekar, R.K. (2022). Steady-State Fluorescence Spectroscopy as a Tool to Monitor Protein/Ligand Interactions. In: Sahoo, H. (eds) Optical Spectroscopic and Microscopic Techniques. Springer, Singapore. https://doi.org/10.1007/978-981-16-4550-1_3

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