title: Protein secondary structure and stability determined by combining exoproteolysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
abstract: In the post-genomic era, several projects focused on the massive experimental resolution of the three-dimensional structures of all the proteins of different organisms have been initiated. Simultaneously, significant progress has been made in the ab initio prediction of protein three-dimensional structure. One of the keys to the success of such a prediction is the use of local information (i.e. secondary structure). Here we describe a new limited proteolysis methodology, based on the use of unspecific exoproteases coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), to map quickly secondary structure elements of a protein from both ends, the N- and C-termini. We show that the proteolytic patterns (mass spectra series) obtained can be interpreted in the light of the conformation and local stability of the analyzed proteins, a direct correlation being observed between the predicted and the experimentally derived protein secondary structure. Further, this methodology can be easily applied to check rapidly the folding state of a protein and characterize mutational effects on protein conformation and stability. Moreover, given global stability information, this methodology allows one to locate the protein regions of increased or decreased conformational stability. All of this can be done with a small fraction of the amount of protein required by most of the other methods for conformational analysis. Thus limited exoproteolysis, together with MALDI-TOF MS, can be a useful tool to achieve quickly the elucidation of protein structure and stability.
authors.affiliation: Institut de Biotecnologia i de Biomedicina, Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona), Spain.
authors.forename: Josep
authors.initials: J
authors.lastname: Villanueva
authors.forename: Virtudes
authors.initials: V
authors.lastname: Villegas
authors.forename: Enrique
authors.initials: E
authors.lastname: Querol
authors.forename: Francesc X
authors.initials: FX
authors.lastname: Avilés
authors.forename: Luis
authors.initials: L
authors.lastname: Serrano
mesh_terms: D002268:Carboxypeptidases / Q000737:chemistry; D043422:Carboxypeptidases A; D010450:Endopeptidases / Q000737:chemistry; D004792:Enzyme Precursors / Q000737:chemistry; D006868:Hydrolysis; D013058:Mass Spectrometry; D009211:Myoglobin / Q000737:chemistry; D010446:Peptide Fragments / Q000032:analysis; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011505:Protein-Tyrosine Kinases / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D004792:Enzyme Precursors; D009211:Myoglobin; D010446:Peptide Fragments; D011506:Proteins; D011505:Protein-Tyrosine Kinases; D002268:Carboxypeptidases; D010450:Endopeptidases; D043422:Carboxypeptidases A