Conformational differences between complexes of elongation factor Tu studied 19F-NMR spectroscopy

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Biophysics

Background:

• Elongation factor Tu (EF-Tu) is crucial for protein synthesis in bacteria.
• EF-Tu undergoes conformational changes upon binding to guanine nucleotides (GDP/GTP) and other factors.
• Understanding these structural dynamics is key to deciphering its function.

Purpose of the Study:

• To investigate the structural environment of tyrosine residues in EF-Tu complexes using 19F-NMR.
• To correlate NMR findings with known X-ray diffraction structures of EF-Tu.
• To elucidate the domain interactions within EF-Tu.GDP and other complexes.

Main Methods:

• Biosynthetic incorporation of 3-fluorotyrosine into EF-Tu from Escherichia coli.
• 19F-NMR spectroscopy of EF-Tu in binary complexes (with GDP, GTP, EF-Ts) and ternary complexes (EF-Tu.GDP.aurodox, EF-Tu.GDP.EF-Ts).
• Comparison of 19F-NMR data with X-ray diffraction structures of EF-Tu.GDP.

Main Results:

• 19F-NMR spectra of EF-Tu.GDP showed two distinct downfield-shifted resonances compared to other complexes.
• These shifts suggest specific tyrosine residues are in a sterically constrained environment in EF-Tu.GDP.
• The observed spectral changes are consistent with close interaction between domain I and domain III in EF-Tu.GDP.

Conclusions:

• The study provides evidence for domain I-domain III interaction in EF-Tu.GDP, mediated by specific tyrosine residues.
• Binding of other factors (GTP, EF-Ts) or structural modifications release these steric restraints.
• 19F-NMR is a valuable tool for probing conformational changes and domain interactions in EF-Tu.