Residue-Level Energetic Analysis of Transient Misfolding in the Whirlin PDZ Tandem Reveals a Possible Connection to an AlphaFold-Predicted Alternative Topology.
- 1Laboratory Affiliated to Istituto Pasteur Italia, Fondazione Cenci Bolognetti, Dipartimento di Scienze Biochimiche "A. Rossi Fanelli", Sapienza Università di Roma, P.le Aldo Moro 5, 00185 Rome, Italy.
- 2Dipartimento di Medicina Clinica, Sanità Pubblica, Scienze Della Vita e Dell'ambiente, Università dell'Aquila, Piazzale Salvatore Tommasi 1, Coppito, 67010 L'Aquila, Italy.
Abstract
Multidomain proteins may populate transient misfolded states that compete with productive folding, but the molecular determinants of these intermediates remain poorly understood. Using the tandem PDZ domains of the scaffolding protein whirlin, in which a misfolded intermediate accumulates only when both domains are denatured, we combined systematic mutagenesis with a thermodynamic framework analogous to double-mutant cycle analysis. Analysis of ~70 mutations identified residues displaying significant non-additive energetic effects, defining an interaction network concentrated within the connecting hairpins, PDZ2, and a few positions at the PDZ1 interface. Interestingly, AlphaFold predicted an alternative domain-swapped topology differing from the experimentally determined structure primarily within these same regions. Although the experimental data do not establish the structure of the misfolded intermediate, this correspondence suggests that the predicted alternative topology may provide a possible structural interpretation of the experimentally identified energetic determinants. Our findings provide a residue-level characterization of the energetic determinants of transient misfolding in a multidomain protein and suggest a possible connection between these determinants and alternative structural arrangements.
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BibTeX
@article{pennacchietti2026residuelevel,
title = {{Residue-Level Energetic Analysis of Transient Misfolding in the Whirlin PDZ Tandem Reveals a Possible Connection to an AlphaFold-Predicted Alternative Topology.}},
author = {Valeria Pennacchietti and Mariana Di Felice and Julian Toso and Eduarda Santos Ventura and Laura Caldarelli and Angelo Toto and Francesca Malagrinò and Stefano Gianni},
journal = {International journal of molecular sciences},
year = {2026},
month = sep,
doi = {10.3390/ijms27198509},
url = {https://europepmc.org/article/MED/42883758},
note = {CC BY},
}
RIS
TY - JOUR
TI - Residue-Level Energetic Analysis of Transient Misfolding in the Whirlin PDZ Tandem Reveals a Possible Connection to an AlphaFold-Predicted Alternative Topology.
AU - Valeria Pennacchietti
AU - Mariana Di Felice
AU - Julian Toso
AU - Eduarda Santos Ventura
AU - Laura Caldarelli
AU - Angelo Toto
AU - Francesca Malagrinò
AU - Stefano Gianni
PY - 2026
DA - 2026/09/24
JO - International journal of molecular sciences
DO - 10.3390/ijms27198509
UR - https://europepmc.org/article/MED/42883758
AB - Multidomain proteins may populate transient misfolded states that compete with productive folding, but the molecular determinants of these intermediates remain poorly understood. Using the tandem PDZ domains of the scaffolding protein whirlin, in which a misfolded intermediate accumulates only when both domains are denatured, we combined systematic mutagenesis with a thermodynamic framework analogous to double-mutant cycle analysis. Analysis of ~70 mutations identified residues displaying significant non-additive energetic effects, defining an interaction network concentrated within the connecting hairpins, PDZ2, and a few positions at the PDZ1 interface. Interestingly, AlphaFold predicted an alternative domain-swapped topology differing from the experimentally determined structure primarily within these same regions. Although the experimental data do not establish the structure of the misfolded intermediate, this correspondence suggests that the predicted alternative topology may provide a possible structural interpretation of the experimentally identified energetic determinants. Our findings provide a residue-level characterization of the energetic determinants of transient misfolding in a multidomain protein and suggest a possible connection between these determinants and alternative structural arrangements.
SN - 1422-0067
ER -