The Interconversion between a Flexible β-Sheet and a Fibril β-Arrangement Constitutes the Main Conformational Event during Misfolding of PSD95-PDZ3 Domain

• Published in: Biophysical journal Vol. 103; no. 4; pp. 738 - 747
• Main Authors: Marin-Argany, Marta, Candel, Adela M., Murciano-Calles, Javier, Martinez, Jose C., Villegas, Sandra
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 22.08.2012; The Biophysical Society
• Subjects: Amino Acid Sequence; Cell Line, Tumor; chemical analysis; cytotoxicity; Fourier transform infrared spectroscopy; heat; Humans; Membrane Proteins - chemistry; Membrane Proteins - toxicity; Models, Molecular; Molecular Sequence Data; nuclear magnetic resonance spectroscopy; PDZ Domains; Protein Folding; Protein Multimerization; Protein Structure, Secondary; Proteins and Nucleic Acids; temperature; transmission electron microscopes; PBS; ThT; PSD95; WL; FTIR; ANS; PDZ; PDZ3; disk-large tumor suppressor; MTT; HSQC; TEM
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1016/j.bpj.2012.07.029

The temperature-induced misfolding pathway of PDZ3, the third PDZ domain of the PSD95 neuronal protein, is populated by a trimeric β-sheet-rich intermediate state that leads to a stepwise and reversible formation of supramacromolecular structures. Using FTIR, we have found that misfolding of this pathway is not due to different ensembles of a variety of precursors, but comes mainly from the interconversion of a flexible β-sheet of the domain to wormlike fibrils. The appearance of the wormlike fibril FTIR component is also accompanied by a slight decrease of the band that corresponds to loops in the native state, whereas the rest of the regular elements of secondary structure are fairly well maintained upon misfolding. Transmission electron microscope micrographs have confirmed the presence of wormlike fibrils upon heating at 60°C, where the trimeric intermediate is maximally populated. Toxicity assays in the human neuroblastoma cell line SH-SY5Y show that cytotoxicity increases as the aggregation pathway proceeds. NMR analysis of chemical shifts as a function of temperature has revealed, as one of the main conformational aspects of such an interconversion at the residue level, that the β-sheet arrangement around strand β3 promotes the change that drives misfolding of the PDZ3 domain.