title: Binding of Amphipathic Cell Penetrating Peptide p28 to Wild Type and Mutated p53 as studied by Raman, Atomic Force and Surface Plasmon Resonance spectroscopies.
abstract: Mutations within the DNA binding domain (DBD) of the tumor suppressor p53 are found in >50% of human cancers and may significantly modify p53 secondary structure impairing its function. p28, an amphipathic cell-penetrating peptide, binds to the DBD through hydrophobic interaction and induces a posttranslational increase in wildtype and mutant p53 restoring functionality. We use mutation analyses to explore which elements of secondary structure may be critical to p28 binding.
  
  
  
  Molecular modeling, Raman spectroscopy, Atomic Force Spectroscopy (AFS) and Surface Plasmon Resonance (SPR) were used to identify which secondary structure of site-directed and naturally occurring mutant DBDs are potentially altered by discrete changes in hydrophobicity and the molecular interaction with p28.
  
  
  
  We show that specific point mutations that alter hydrophobicity within non-mutable and mutable regions of the p53 DBD alter specific secondary structures. The affinity of p28 was positively correlated with the β-sheet content of a mutant DBD, and reduced by an increase in unstructured or random coil that resulted from a loss in hydrophobicity and redistribution of surface charge.
  
  
  
  These results help refine our knowledge of how mutations within p53-DBD alter secondary structure and provide insight on how potential structural alterations in p28 or similar molecules improve their ability to restore p53 function.
  
  
  
  Raman spectroscopy, AFS, SPR and computational modeling are useful approaches to characterize how mutations within the p53DBD potentially affect secondary structure and identify those structural elements prone to influence the binding affinity of agents designed to increase the functionality of p53.
authors.affiliation: Biophysics and Nanoscience Centre, DEB, Università della Tuscia, Viterbo, Italy; Department of Science, University Roma Tre, Rome, Italy.
authors.forename: Sara
authors.initials: S
authors.lastname: Signorelli
authors.affiliation: Biophysics and Nanoscience Centre, DEB, Università della Tuscia, Viterbo, Italy.
authors.forename: Simona
authors.initials: S
authors.lastname: Santini
authors.affiliation: Department of Surgery, Division of Surgical Oncology, University of Illinois College of Medicine, Chicago, IL, USA.
authors.forename: Tohru
authors.initials: T
authors.lastname: Yamada
authors.affiliation: Biophysics and Nanoscience Centre, DEB, Università della Tuscia, Viterbo, Italy. Electronic address: bizzarri@unitus.it.
authors.forename: Anna Rita
authors.initials: AR
authors.lastname: Bizzarri
authors.affiliation: Department of Surgery, Division of Surgical Oncology, University of Illinois College of Medicine, Chicago, IL, USA.
authors.forename: Craig W
authors.initials: CW
authors.lastname: Beattie
authors.affiliation: Biophysics and Nanoscience Centre, DEB, Università della Tuscia, Viterbo, Italy.
authors.forename: Salvatore
authors.initials: S
authors.lastname: Cannistraro
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D045744:Cell Line, Tumor; D057846:Cell-Penetrating Peptides / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D018625:Microscopy, Atomic Force / Q000379:methods; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D009154:Mutation / Q000235:genetics; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017433:Protein Structure, Secondary; D020349:Surface Plasmon Resonance / Q000379:methods; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
chemical_list: D057846:Cell-Penetrating Peptides; D004268:DNA-Binding Proteins; D010446:Peptide Fragments; D016159:Tumor Suppressor Protein p53
keywords: Atomic Force Spectroscopy; Cell penetrating peptide; Docking; Mutations; Raman spectroscopy; p53