Proximity-Triggered Covalent Stabilization of Low-Affinity Protein Complexes In Vitro and In Vivo
Standard
Proximity-Triggered Covalent Stabilization of Low-Affinity Protein Complexes In Vitro and In Vivo. / Cigler, Marko; Müller, Thorsten G; Horn-Ghetko, Daniel; von Wrisberg, Marie-Kristin; Fottner, Maximilian; Goody, Roger S; Itzen, Aymelt; Lang, Kathrin.
in: ANGEW CHEM INT EDIT, Jahrgang 56, Nr. 49, 04.12.2017, S. 15737-15741.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
Harvard
APA
Vancouver
Bibtex
}
RIS
TY - JOUR
T1 - Proximity-Triggered Covalent Stabilization of Low-Affinity Protein Complexes In Vitro and In Vivo
AU - Cigler, Marko
AU - Müller, Thorsten G
AU - Horn-Ghetko, Daniel
AU - von Wrisberg, Marie-Kristin
AU - Fottner, Maximilian
AU - Goody, Roger S
AU - Itzen, Aymelt
AU - Lang, Kathrin
N1 - © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
PY - 2017/12/4
Y1 - 2017/12/4
N2 - The characterization of low-affinity protein complexes is challenging due to their dynamic nature. Here, we present a method to stabilize transient protein complexes in vivo by generating a covalent and conformationally flexible bridge between the interaction partners. A highly active pyrrolysyl tRNA synthetase mutant directs the incorporation of unnatural amino acids bearing bromoalkyl moieties (BrCnK) into proteins. We demonstrate for the first time that low-affinity protein complexes between BrCnK-containing proteins and their binding partners can be stabilized in vivo in bacterial and mammalian cells. Using this approach, we determined the crystal structure of a transient GDP-bound complex between a small G-protein and its nucleotide exchange factor. We envision that this approach will prove valuable as a general tool for validating and characterizing protein-protein interactions in vitro and in vivo.
AB - The characterization of low-affinity protein complexes is challenging due to their dynamic nature. Here, we present a method to stabilize transient protein complexes in vivo by generating a covalent and conformationally flexible bridge between the interaction partners. A highly active pyrrolysyl tRNA synthetase mutant directs the incorporation of unnatural amino acids bearing bromoalkyl moieties (BrCnK) into proteins. We demonstrate for the first time that low-affinity protein complexes between BrCnK-containing proteins and their binding partners can be stabilized in vivo in bacterial and mammalian cells. Using this approach, we determined the crystal structure of a transient GDP-bound complex between a small G-protein and its nucleotide exchange factor. We envision that this approach will prove valuable as a general tool for validating and characterizing protein-protein interactions in vitro and in vivo.
KW - Amino Acids
KW - Amino Acyl-tRNA Synthetases
KW - GTP-Binding Protein Regulators
KW - GTP-Binding Proteins
KW - Green Fluorescent Proteins
KW - HEK293 Cells
KW - Humans
KW - Models, Molecular
KW - Mutation
KW - Protein Binding
KW - Protein Stability
KW - Journal Article
KW - Research Support, Non-U.S. Gov't
U2 - 10.1002/anie.201706927
DO - 10.1002/anie.201706927
M3 - SCORING: Journal article
C2 - 28960788
VL - 56
SP - 15737
EP - 15741
JO - ANGEW CHEM INT EDIT
JF - ANGEW CHEM INT EDIT
SN - 1433-7851
IS - 49
ER -