Acceleration of protein backbone NMR assignment by combinatorial labeling: Application to a small molecule binding...

Area of Science:

• Biochemistry
• Structural Biology
• Nuclear Magnetic Resonance (NMR) Spectroscopy

Background:

• Stable isotope labeling is crucial in protein NMR for overcoming signal overlap and sensitivity issues.
• Selective labeling strategies are essential for detailed structural and functional analyses of proteins.

Purpose of the Study:

• To develop and optimize a combinatorial selective labeling scheme for efficient backbone assignment of human cyclophilin D.
• To apply this labeling strategy in conjunction with NMR experiments to study inhibitor binding.

Main Methods:

• Utilized a cell-free expression system for combinatorial labeling with various ¹⁵N and ¹³C isotopes.
• Optimized a labeling scheme involving ¹⁵N, 1-¹³C, 2-¹³C, fully ¹⁵N/¹³C, and unlabeled amino acids.
• Employed time-shared triple-resonance NMR experiments for rapid and comprehensive backbone assignment.

Main Results:

• Achieved maximum assignment information from just three samples using the optimized labeling scheme.
• Enabled unambiguous assignment of unique and ambiguous amino acid pairs.
• Provided information on all 19 non-proline amino acid types within the protein.

Conclusions:

• The developed combinatorial selective labeling scheme is highly efficient for protein backbone assignment.
• This method is well-suited for de novo resonance assignments in binding studies, particularly when sensitivity is a limitation.
• Facilitates structural insights into protein-inhibitor interactions using NMR spectroscopy.