TY - JOUR
T1 - Hyperstable de novo protein with a dimeric bisecting topology
AU - Kimura, Naoya
AU - Mochizuki, Kenji
AU - Umezawa, Koji
AU - Hecht, Michael H.
AU - Arai, Ryoichi
N1 - Funding Information:
We sincerely thank Prof. Akihiko Yamagishi, Dr. Sota Yagi, and Mr. Takahiro Sasamoto at Tokyo University of Pharmacy and Life Sciences and Prof. Satoshi Akanuma at Waseda University for help in CD experiments; Dr. Takahiro Kosugi, Dr. Rie Koga, and Dr. Nobuyasu Koga at Institute for Molecular Science (IMS) for assistance in SEC–MALS and CD experiments; Dr. Naoya Kobayashi at IMS, and Prof. Takaaki Sato, Mr. Kouichi Inano, and Dr. Keiichi Yanase at Shinshu University for help in SAXS analysis; Mr. Yusuke Koyanagi at Shinshu University and Dr. Keitaro Yamashita at University of Tokyo for help in installing MR-Rosetta. The synchrotron X-ray experiments were performed at Photon Factory (PF), KEK, under the approval of PF program advisory committee (Proposal No. 2014G111, 2014G673, 2016G153, 2016G606, 2016G617, 2018G634, and 2018G636). We thank the beamline scientists and staff at PF, KEK. This work was supported by JSPS KAKENHI Grant numbers JP16K05841, JP16H00761, JP24780097, JP17KK0104, and JP19H02522. This work was also supported by Joint Research program of IMS and JSPS Invitational Fellowship.
Publisher Copyright:
© 2020 American Chemical Society.
PY - 2020/2/21
Y1 - 2020/2/21
N2 - Recently, we designed and assembled protein nanobuilding blocks (PN-Blocks) from an intermolecularly folded dimeric de novo protein called WA20. Using this dimeric 4-helix bundle, we constructed a series of self-assembling supramolecular nanostructures including polyhedra and chain-type complexes. Here we describe the stabilization of WA20 by designing mutations that stabilize the helices and hydrophobic core. The redesigned proteins denature with substantially higher midpoints, with the most stable variant, called Super WA20 (SUWA), displaying an extremely high midpoint (Tm = 122 °C), much higher than the Tm of WA20 (75 °C). The crystal structure of SUWA reveals an intermolecularly folded dimer with bisecting U topology, similar to the parental WA20 structure, with two long α-helices of a protomer intertwined with the helices of another protomer. Molecular dynamics simulations demonstrate that the redesigned hydrophobic core in the center of SUWA significantly suppresses the deformation of helices observed in the same region of WA20, suggesting this is a critical factor stabilizing the SUWA structure. This hyperstable de novo protein is expected to be useful as nanoscale pillars of PN-Block components in new types of self-assembling nanoarchitectures.
AB - Recently, we designed and assembled protein nanobuilding blocks (PN-Blocks) from an intermolecularly folded dimeric de novo protein called WA20. Using this dimeric 4-helix bundle, we constructed a series of self-assembling supramolecular nanostructures including polyhedra and chain-type complexes. Here we describe the stabilization of WA20 by designing mutations that stabilize the helices and hydrophobic core. The redesigned proteins denature with substantially higher midpoints, with the most stable variant, called Super WA20 (SUWA), displaying an extremely high midpoint (Tm = 122 °C), much higher than the Tm of WA20 (75 °C). The crystal structure of SUWA reveals an intermolecularly folded dimer with bisecting U topology, similar to the parental WA20 structure, with two long α-helices of a protomer intertwined with the helices of another protomer. Molecular dynamics simulations demonstrate that the redesigned hydrophobic core in the center of SUWA significantly suppresses the deformation of helices observed in the same region of WA20, suggesting this is a critical factor stabilizing the SUWA structure. This hyperstable de novo protein is expected to be useful as nanoscale pillars of PN-Block components in new types of self-assembling nanoarchitectures.
KW - 4-helix bundle
KW - Binary pattern
KW - Bisecting U topology
KW - De novo protein
KW - Protein nanobuilding block
KW - Protein stabilization
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U2 - 10.1021/acssynbio.9b00501
DO - 10.1021/acssynbio.9b00501
M3 - Article
C2 - 31951376
AN - SCOPUS:85081035859
SN - 2161-5063
VL - 9
SP - 254
EP - 259
JO - ACS Synthetic Biology
JF - ACS Synthetic Biology
IS - 2
ER -