Abstract
Raman spectroscopy, which is a suitable tool to elucidate the structural properties of intrinsically disordered proteins, was applied to investigate the changes in both the structure and the conformational heterogeneity of the DNA-binding domain (DBD) belonging to the intrinsically disordered protein p53 upon its binding to Azurin, an electron-transfer anticancer protein from Pseudomonas aeruginosa. The Raman spectra of the DBD and Azurin, isolated in solution or forming a complex, were analyzed by a combined analysis based on peak inspection, band convolution, and principal component analysis (PCA). In particular, our attention was focused on the Raman peaks of Tyrosine and Tryptophan residues, which are diagnostic markers of protein side chain environment, and on the Amide I band, of which the deconvolution allows us to extract information about α-helix, β-sheet, and random coil contents. The results show an increase of the secondary structure content of DBD concomitantly with a decrease of its conformational heterogeneity upon its binding to Azurin. These findings suggest an Azurin-induced conformational change of DBD structure with possible implications for p53 functionality.
Highlights
Raman spectroscopy, which is a suitable tool to elucidate the structural properties of intrinsically disordered proteins, was applied to investigate the changes in both the structure and the conformational heterogeneity of the DNA-binding domain (DBD) belonging to the intrinsically disordered protein p53 upon its binding to Azurin, an electron-transfer anticancer protein from Pseudomonas aeruginosa
Introduction p53 is an important tumor suppressor protein working as a central hub in a complex interaction network in which it regulates numerous cellular processes, including cell cycle progression, apoptosis induction, and DNA repair [1,2]. p53 is a member of the important class of intrinsically disordered proteins (IDPs), possessing both structured and disordered domains under physiological conditions and different conformations coexisting in solution [3]
Each monomer of p53 consists of an N-terminal transactivation domain (NTD), a C-terminal domain (CTD), and a core DNA-binding domain (DBD) [7,8,9,10] The presence of unstructured portions allows p53 to adopt widely different conformations, which are at the basis of a vast repertoire of available interactions to different biological partners [11]
Summary
Raman spectroscopy, which is a suitable tool to elucidate the structural properties of intrinsically disordered proteins, was applied to investigate the changes in both the structure and the conformational heterogeneity of the DNA-binding domain (DBD) belonging to the intrinsically disordered protein p53 upon its binding to Azurin, an electron-transfer anticancer protein from Pseudomonas aeruginosa. No experimental evidences on possible structural alterations of p53 upon its binding to AZ are so far available [3] In this respect, Raman spectroscopy represents a suitable approach to extract information about the secondary structure of proteins as well as to probe their conformational heterogeneity, including IDPs [22]. To such an aim, we have focused on an accurate inspection of the Fermi doublets relative to Tyrosine (830 and 850 cm−1; Tyr) and of Tryptophan peaks (1340 and 1360 cm−1), with these Raman signals having been recognized as suitable diagnostic markers of protein side chain environment [25,26]. The Raman spectra have been analyzed by applying principal component analysis (PCA), which performs a dimensionality reduction of the spectra, allowing a revelation of the differences between the complex Raman spectra of the samples and helping to understand the principal factors affecting the spectral variation [33]
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