title: Multiple conformations of full-length p53 detected with single-molecule fluorescence resonance energy transfer.
abstract: The tumor suppressor p53 is a member of the emerging class of proteins that have both folded and intrinsically disordered domains, which are a challenge to structural biology. Its N-terminal domain (NTD) is linked to a folded core domain, which has a disordered link to the folded tetramerization domain, which is followed by a disordered C-terminal domain. The quaternary structure of human p53 has been solved by a combination of NMR spectroscopy, electron microscopy, and small-angle X-ray scattering (SAXS), and the NTD ensemble structure has been solved by NMR and SAXS. The murine p53 is reported to have a different quaternary structure, with the N and C termini interacting. Here, we used single-molecule FRET (SM-FRET) and ensemble FRET to investigate the conformational dynamics of the NTD of p53 in isolation and in the context of tetrameric full-length p53 (flp53). Our results showed that the isolated NTD was extended in solution with a strong preference for residues 66-86 forming a polyproline II conformation. The NTD associated weakly with the DNA binding domain of p53, but not the C termini. We detected multiple conformations in flp53 that were likely to result from the interactions of NTD with the DNA binding domain of each monomeric p53. Overall, the SM-FRET results, in addition to corroborating the previous ensemble findings, enabled the identification of the existence of multiple conformations of p53, which are often averaged and neglected in conventional ensemble techniques. Our study exemplifies the usefulness of SM-FRET in exploring the dynamic landscape of multimeric proteins that contain regions of unstructured domains.
authors.affiliation: Medical Research Council Centre for Protein Engineering, Hills Road, Cambridge CB2 0QH, United Kingdom.
authors.forename: Fang
authors.initials: F
authors.lastname: Huang
authors.forename: Sridharan
authors.initials: S
authors.lastname: Rajagopalan
authors.forename: Giovanni
authors.initials: G
authors.lastname: Settanni
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Marsh
authors.forename: Daven A
authors.initials: DA
authors.lastname: Armoogum
authors.forename: Nick
authors.initials: N
authors.lastname: Nicolaou
authors.forename: Angus J
authors.initials: AJ
authors.lastname: Bain
authors.forename: Eitan
authors.initials: E
authors.lastname: Lerner
authors.forename: Elisha
authors.initials: E
authors.lastname: Haas
authors.forename: Liming
authors.initials: L
authors.lastname: Ying
authors.forename: Alan R
authors.initials: AR
authors.lastname: Fersht
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D004058:Diffusion; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D053838:Scattering, Small Angle; D013997:Time Factors; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000378:metabolism*; D014961:X-Ray Diffraction
chemical_list: D000596:Amino Acids; D050505:Mutant Proteins; D021122:Protein Subunits; D016159:Tumor Suppressor Protein p53