Monitoring disappearance of monomers and generation of resistance to proteolysis during the formation of the activation domain of human procarboxypeptidase A2 (ADA2h) amyloid fibrils by matrix-assisted laser-desorption ionization-time-of-flight-MS.
The term 'amyloidosis' is used to represent a group of protein misfolding diseases characterized by the polymerization of normally innocuous and soluble proteins or peptides into insoluble proteinaceous deposits. One of the several questions that remain unclear regarding the process of amyloid fibril formation is related to the status of the protein when such a process begins. Protein engineering is one of the selected approaches to study amyloidosis. Characterization of many variants of a protein can give information about why a soluble protein aggregates to form fibrils. In the present study, we report information on the conformational changes that precede the formation of fibrils, monitored by the complementary use of exoproteolysis and matrix-assisted laser-desorption ionization-time-of-flight-MS. This is a novel application of an easy and fast approach. In addition, we used it to evaluate the ability of the model protein ADA2h (activation domain of human procarboxypeptidase A2) and their mutants to generate amyloid fibrils. It could be a useful test to screen protein variants and to study to what extent some physicochemical parameters affect fibrillogenesis.
Villanueva
Josep
J
Institut de Biotecnologia i Biomedicina, and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain.
Villegas
Virtudes
V
Querol
Enrique
E
Avilés
Francesc X
FX
Serrano
Luis
L
D000682:Amyloid / Q000737:chemistry / Q000378:metabolism*; D002268:Carboxypeptidases / Q000737:chemistry / Q000378:metabolism*; D043422:Carboxypeptidases A; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation / Q000502:physiology; D004792:Enzyme Precursors / Q000737:chemistry / Q000378:metabolism*; D020689:Exopeptidases / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D010446:Peptide Fragments / Q000378:metabolism; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D000682:Amyloid; D004792:Enzyme Precursors; D010446:Peptide Fragments; D002268:Carboxypeptidases; D010450:Endopeptidases; D020689:Exopeptidases; D043422:Carboxypeptidases A