<p>Background: Conformational flexibility of proteins remains as one of the major events in protein-protein/DNA/ligand/small molecule binding to achieve its biological function in the cell. The availability of high-resolution structures of protein complexes is a valuable resource for researchers to understand the mechanisms behind such interactions and it is found that the flexibility of amino acid residues at binding sites is crucial for many important functions in the cell.<br /> Methods: In this article, our statistical method (PreFRP) developed based on fluctuating amino acid residues and various amino acid indices related to flexibility/rigidity were used to study the importance of fluctuating amino acid residues in thermonuc-leases from pathogenic bacteria, cell penetrating peptides and intrinsically disordered proteins responsible for many neural disorders.&nbsp;<br /> Results: The results from our analysis reveal the importance of fluctuating amino acid residues in folding and binding of proteins. The role of moderate and high fluctuating residues in themonucleases, cell penetrating peptide and disordered regions are discussed in detail.<br /> Conclusion: Therefore, our analysis will help in understanding the importance of fluc-tuating amino acid residues in proteins which undergo a conformation change phenomenon.</p>