Structural Characterization of the Intrinsically Disordered Protein p53 Using Raman Spectroscopy

Abstract

The intrinsically disordered protein p53 has attracted a strong interest for its important role in genome safeguarding and potential therapeutic applications. However, its disordered character makes difficult a full characterization of p53 structural architecture. A deep knowledge of p53 structural motifs could significantly help the understanding of its functional properties, in connection with its complex binding network. We have applied Raman spectroscopy to investigate the structural composition and the conformational heterogeneity of both full-length p53 and its DNA binding domain (DBD), in different solvent environments. In particular, a careful analysis of the Amide I Raman band, which is highly sensitive to protein secondary structure elements such as α-helices, β-sheets and random coils, has revealed the presence of extended random coils in p53 and predominant β-sheet regions in its DBD. In addition, this analysis has allowed us to explore the ensemble of interchanging conformations in both p53 and its DBD, highlighting a higher conformational heterogeneity in p53 than in its DBD. Furthermore, by applying a principal components analysis, we have identified the principal spectral markers in both p53 and DBD samples. The combination of the two approaches could be insightful for the study of intrinsically disordered proteins, by offering increased versatility and wide application as a label-free, real-time and non-invasive detection method.

Characterization of wild‐type and mutant p53 protein by Raman spectroscopy and multivariate methods

“…Figure shows the corresponding Raman spectrum from each specimen of p53 protein. Usually, the fingerprint region between 400 and 1,800 cm −1 provides the most useful information about the protein structure so that we extend the measurements to 300–2,000 cm −1 to observe a wider region . As expected, the vibrational spectra contain a complex set of peaks and overlapping bands, which cannot be used directly for structural determination; however, several studies have been carried out to investigate the Raman spectrum of individual amino acids, dipeptides, and tripeptides, whose results are useful for, based on frequency coincidences, assigning vibrations to the most prominent bands in the protein spectrum .…”

“…As expected, the vibrational spectra contain a complex set of peaks and overlapping bands, which cannot be used directly for structural determination; however, several studies have been carried out to investigate the Raman spectrum of individual amino acids, dipeptides, and tripeptides, whose results are useful for, based on frequency coincidences, assigning vibrations to the most prominent bands in the protein spectrum . Features observed in the wt p53 spectrum are closer with those previously reported by Signorelli et al, but in addition, we observed two prominent peaks in 416 and 480 cm −1 that could be assigned to the symmetric stretching band and are present in the mutants as well. For all p53 types, the dominant peak at 848 cm −1 and the shoulder in 825 cm −1 correspond to the scattering of aromatic amino acid Tyr.…”

“…The three mutants present a shoulder at 764 cm −1 for Trp, two sharp peaks at 1,258 and 1,328 cm −1 assigned to CH 2 groups, and the vibration in 1,131 cm −1 for Leu. For the mutant 344, Phe is well visible at 620 cm −1 …”

“…Usually, the fingerprint region between 400 and 1,800 cm −1 provides the most useful information about the protein structure so that we extend the measurements to 300-2,000 cm −1 to observe a wider region. [33,34] As expected, the vibrational spectra contain a complex set of peaks and overlapping bands, which cannot be used directly for structural determination; however, several studies have been carried out to investigate the Raman spectrum of individual amino acids, dipeptides, and tripeptides, whose results are useful for, based on frequency coincidences, assigning vibrations to the most prominent bands in the protein spectrum. [33,35] Features observed in the wt p53 spectrum are closer with those previously reported by Signorelli et al, [34] but in addition, we observed two prominent peaks in 416 and 480 cm −1 that could be assigned to the symmetric stretching band and are present in the mutants as well.…”

Recent advances in hybrid measurement methods based on atomic force microscopy and surface sensitive measurement techniques

“…gemstone identication), 227 biology (e.g. secondary structure of polypeptides/proteins, phospholipids), [228][229][230][231] material science 232,233 (e.g. semiconductors, carbon materials and polymers) and life science (e.g.…”