Reverse Engineering Analysis of the High-Temperature Reversible Oligomerization and Amyloidogenicity of PSD95-PDZ3 Onchaiya, Sawaros Martínez Herrerías, José Cristóbal High-temperature reversible oligomerization Amyloidogenicity Oligomeric interface residues Thermal denaturation Mutational analysis This research was funded by a MEXT scholarship to SO, a JSPS grant-in-aid for scientific research (KAKENHI: 18H02385, 21K05288, and 21K15049) and the TUAT's Institute of Global Innovation Research. PSD95-PDZ3, the third PDZ domain of the post-synaptic density-95 protein (MW 11 kDa), undergoes a peculiar three-state thermal denaturation (N <-> I-n <-> D) and is amyloidogenic. PSD95-PDZ3 in the intermediate state (I) is reversibly oligomerized (RO: Reversible oligomerization). We previously reported a point mutation (F340A) that inhibits both ROs and amyloidogenesis and constructed the PDZ3-F340A variant. Here, we "reverse engineered" PDZ3-F340A for inducing high-temperature RO and amyloidogenesis. We produced three variants (R309L, E310L, and N326L), where we individually mutated hydrophilic residues exposed at the surface of the monomeric PDZ3-F340A but buried in the tetrameric crystal structure to a hydrophobic leucine. Differential scanning calorimetry indicated that two of the designed variants (PDZ3-F340A/R309L and E310L) denatured according to the two-state model. On the other hand, PDZ3-F340A/N326L denatured according to a three-state model and produced high-temperature ROs. The secondary structures of PDZ3-F340A/N326L and PDZ3-wt in the RO state were unfolded according to circular dichroism and differential scanning calorimetry. Furthermore, PDZ3-F340A/N326L was amyloidogenic as assessed by Thioflavin T fluorescence. Altogether, these results demonstrate that a single amino acid mutation can trigger the formation of high-temperature RO and concurrent amyloidogenesis. 2022-06-02T06:33:27Z 2022-06-02T06:33:27Z 2022-04-28 info:eu-repo/semantics/article Onchaiya, S... [et al.]. Reverse Engineering Analysis of the High-Temperature Reversible Oligomerization and Amyloidogenicity of PSD95-PDZ3. Molecules 2022, 27, 2813. [https://doi.org/10.3390/molecules27092813] http://hdl.handle.net/10481/75185 10.3390/molecules27092813 eng http://creativecommons.org/licenses/by/3.0/es/ info:eu-repo/semantics/openAccess Atribución 3.0 España MDPI