Structural and Stability Analysis of de Novo Designed Proteins Incorporating a Plant Self‐Incompatibility Motif
4 affiliations
- 1Department of Biotechnology, Graduate School of Agricultural and Life Sciences The University of Tokyo Tokyo Japan
- 2Department of Basic Biology, Cellular and Structural Physiology Institute Nagoya University Nagoya Aichi Japan
- 3Medical Research Laboratory, Institute of Integrated Research Institute of Science Tokyo Tokyo Japan
- 4Collaborative Research Institute for Innovative Microbiology The University of Tokyo Tokyo Japan
Abstract
Artificial protein design enables the creation of stable scaffolds beyond those evolved in nature. Incorporation of native functional motifs into de novo scaffolds provides a promising strategy to mimic natural interactions while altering structural frameworks. Here, we designed de novo S‐locus Protein 11 (SP11)‐like proteins by incorporating a key feature of the Brassica pollen determinant SP11, which mediates self‐incompatibility through specific interaction with the pistil determinant S receptor kinase (SRK). The designed proteins included the six‐amino‐acid SRK‐binding motif from native SP11 but lacked all disulfide bonds characteristic of the plant defensin‐like fold. Three variants (SP11‐A, SP11‐B, SP11‐C) were designed using Rosetta and ProteinMPNN sequence optimization and expressed in Escherichia coli . SP11‐A and SP11‐B were purified and analyzed by circular dichroism spectroscopy. SP11‐A exhibited exceptional thermal and chemical stability, demonstrating the robustness of the artificial scaffold, while SP11‐B had lower stability and displayed biphasic chemical unfolding behavior. The crystal structures of SP11‐A in two space groups were determined at 1.50 and 2.10 Å resolution. Notably, the region derived from the native SP11 sequence exhibited two alternative main chain conformations, indicating that incorporation of the natural motif introduced local conformational frustration. Molecular dynamics analysis suggested that the two alternative main chain conformations remain kinetically stable within the 100 ns simulation timescale, without direct interconversion. Together, these results provide insight into how the incorporation of native motifs can influence local structure while preserving global scaffold stability in de novo protein design.
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BibTeX
@article{miyoshi2026structural,
title = {{Structural and Stability Analysis of de Novo Designed Proteins Incorporating a Plant Self‐Incompatibility Motif}},
author = {Hakuto Miyoshi and Koya Sakuma and Yoshitaka Moriwaki and Naoki Aoyama and Toma Kashima and Tohru Terada and Akimasa Miyanaga and Shinya Fushinobu},
journal = {Proteins},
year = {2026},
month = jul,
doi = {10.1002/prot.70162},
url = {https://doi.org/10.1002/prot.70162},
note = {CC BY-NC-ND 4.0},
}
RIS
TY - JOUR
TI - Structural and Stability Analysis of de Novo Designed Proteins Incorporating a Plant Self‐Incompatibility Motif
AU - Hakuto Miyoshi
AU - Koya Sakuma
AU - Yoshitaka Moriwaki
AU - Naoki Aoyama
AU - Toma Kashima
AU - Tohru Terada
AU - Akimasa Miyanaga
AU - Shinya Fushinobu
PY - 2026
DA - 2026/07/24
JO - Proteins
DO - 10.1002/prot.70162
UR - https://doi.org/10.1002/prot.70162
AB - Artificial protein design enables the creation of stable scaffolds beyond those evolved in nature. Incorporation of native functional motifs into de novo scaffolds provides a promising strategy to mimic natural interactions while altering structural frameworks. Here, we designed de novo S‐locus Protein 11 (SP11)‐like proteins by incorporating a key feature of the Brassica pollen determinant SP11, which mediates self‐incompatibility through specific interaction with the pistil determinant S receptor kinase (SRK). The designed proteins included the six‐amino‐acid SRK‐binding motif from native SP11 but lacked all disulfide bonds characteristic of the plant defensin‐like fold. Three variants (SP11‐A, SP11‐B, SP11‐C) were designed using Rosetta and ProteinMPNN sequence optimization and expressed in Escherichia coli . SP11‐A and SP11‐B were purified and analyzed by circular dichroism spectroscopy. SP11‐A exhibited exceptional thermal and chemical stability, demonstrating the robustness of the artificial scaffold, while SP11‐B had lower stability and displayed biphasic chemical unfolding behavior. The crystal structures of SP11‐A in two space groups were determined at 1.50 and 2.10 Å resolution. Notably, the region derived from the native SP11 sequence exhibited two alternative main chain conformations, indicating that incorporation of the natural motif introduced local conformational frustration. Molecular dynamics analysis suggested that the two alternative main chain conformations remain kinetically stable within the 100 ns simulation timescale, without direct interconversion. Together, these results provide insight into how the incorporation of native motifs can influence local structure while preserving global scaffold stability in de novo protein design.
SN - 0887-3585
SN - 1097-0134
ER -