Intramolecular movements in EF-G, trapped at different stages in its GTP hydrolytic cycle, probed by FRET.
Elongation factor G (EF-G) is one of several GTP hydrolytic proteins (GTPases) that cycles repeatedly on and off the ribosome during protein synthesis in bacterial cells. In the functional cycle of EF-G, hydrolysis of guanosine 5'-triphosphate (GTP) is coupled to tRNA-mRNA translocation in ribosomes. GTP hydrolysis induces conformational rearrangements in two switch elements in the G domain of EF-G and other GTPases. These switch elements are thought to initiate the cascade of events that lead to translocation and EF-G cycling between ribosomes. To further define the coupling mechanism, we developed a new fluorescent approach that can detect intramolecular movements in EF-G. We attached a fluorescent probe to the switch I element (sw1) of Escherichia coli EF-G. We monitored the position of the sw1 probe, relative to another fluorescent probe anchored to the GTP substrate or product, by measuring the distance-dependent, Förster resonance energy transfer between the two probes. By analyzing EF-G trapped at five different functional states in its cycle, we could infer the cyclical movements of sw1 within EF-G. Our results provide evidence for conformational changes in sw1, which help to drive the unidirectional EF-G cycle during protein synthesis. More generally, our approach might also serve to define the conformational dynamics of other GTPases with their cellular receptors.
Nguyen
Boray
B
Department of Biochemistry, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
Ticu
Cristina
C
Wilson
Kevin S
KS
D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006868:Hydrolysis; D056004:Molecular Dynamics Simulation; D009068:Movement; D020653:Peptide Elongation Factor G / Q000737:chemistry* / Q000378:metabolism; D014176:Protein Biosynthesis; D011487:Protein Conformation; D012270:Ribosomes
D029968:Escherichia coli Proteins; D020653:Peptide Elongation Factor G; D006160:Guanosine Triphosphate