Telluromethionine in structural biochemistry

Area of Science:

• Biochemistry
• Structural Biology
• Biophysics

Background:

• Solving the phase problem in macromolecular crystallography requires suitable heavy-atom derivatives.
• Internal labeling of proteins offers a valuable tool for crystallographic phasing and spectroscopic studies.

Purpose of the Study:

• To demonstrate the partial incorporation of telluromethionine into E. coli dihydrofolate reductase (DHFR) via protein biosynthesis.
• To evaluate the impact of telluromethionine incorporation on DHFR activity and substrate binding.
• To establish telluromethionine as a useful tool for both X-ray crystallography and NMR spectroscopy.

Main Methods:

• Utilizing protein biosynthesis for site-specific incorporation of telluromethionine into E. coli dihydrofolate reductase.
• Assessing enzyme activity through specific activity measurements.
• Determining substrate binding affinity using binding ratio analysis with methotrexate.

Main Results:

• Partial incorporation of telluromethionine into E. coli DHFR was achieved without apparent perturbations to enzyme activity or substrate binding.
• Enzymes containing two moles of telluromethionine exhibited a specific activity of 42 units/mg.
• A 1:1 binding ratio with methotrexate was observed for the telluromethionine-containing DHFR.

Conclusions:

• Telluromethionine incorporation via protein biosynthesis is a viable method for generating heavy-atom derivatives for crystallography.
• Telluromethionine serves as an effective internal probe for nuclear magnetic resonance (NMR) studies, enabling structure/function investigations.
• This approach provides a convenient phasing vehicle for X-ray crystallography and a tool for studying protein structure-function relationships.