Fusion of DARPin to Aldolase Enables Visualization of Small Protein by Cryo-EM.
Animals
Binding Sites
Cloning, Molecular
Cryoelectron Microscopy
/ methods
Escherichia coli
/ genetics
Fructose-Bisphosphate Aldolase
/ chemistry
Gene Expression
Genetic Vectors
/ chemistry
Green Fluorescent Proteins
/ chemistry
Humans
Models, Molecular
Muscle Proteins
/ chemistry
Protein Binding
Protein Conformation, alpha-Helical
Protein Conformation, beta-Strand
Protein Engineering
/ methods
Protein Interaction Domains and Motifs
Protein Multimerization
Rabbits
Recombinant Fusion Proteins
/ chemistry
Recombinant Proteins
/ chemistry
Single Molecule Imaging
Single-Chain Antibodies
/ chemistry
beta-Galactosidase
/ chemistry
CryoEM
DARPin
Electron cryo-microscopy
Single-particle analysis
aldolase
artificial protein
cryo-electron microscopy
platform
protein design
Journal
Structure (London, England : 1993)
ISSN: 1878-4186
Titre abrégé: Structure
Pays: United States
ID NLM: 101087697
Informations de publication
Date de publication:
02 07 2019
02 07 2019
Historique:
received:
02
12
2018
revised:
04
03
2019
accepted:
05
04
2019
pubmed:
14
5
2019
medline:
28
4
2020
entrez:
14
5
2019
Statut:
ppublish
Résumé
Solving protein structures by single-particle cryoelectron microscopy (cryo-EM) has become a crucial tool in structural biology. While exciting progress is being made toward the visualization of small macromolecules, the median protein size in both eukaryotes and bacteria is still beyond the reach of cryo-EM. To overcome this problem, we implemented a platform strategy in which a small protein target was rigidly attached to a large, symmetric base via a selectable adapter. Of our seven designs, the best construct used a designed ankyrin repeat protein (DARPin) rigidly fused to tetrameric rabbit muscle aldolase through a helical linker. The DARPin retained its ability to bind its target: GFP. We solved the structure of this complex to 3.0 Å resolution overall, with 5-8 Å resolution in the GFP region. As flexibility in the DARPin position limited the overall resolution of the target, we describe strategies to rigidify this element.
Identifiants
pubmed: 31080120
pii: S0969-2126(19)30125-X
doi: 10.1016/j.str.2019.04.003
pmc: PMC6610650
mid: NIHMS1528032
pii:
doi:
Substances chimiques
Muscle Proteins
0
Recombinant Fusion Proteins
0
Recombinant Proteins
0
Single-Chain Antibodies
0
Green Fluorescent Proteins
147336-22-9
beta-Galactosidase
EC 3.2.1.23
Fructose-Bisphosphate Aldolase
EC 4.1.2.13
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
1148-1155.e3Subventions
Organisme : NIGMS NIH HHS
ID : P50 GM082545
Pays : United States
Organisme : Howard Hughes Medical Institute
Pays : United States
Informations de copyright
Copyright © 2019 Elsevier Ltd. All rights reserved.
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