APSY-NMR for protein backbone assignment in high-throughput structural biology

Area of Science:

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

Background:

• The Protein Structure Initiative (PSI) and Joint Center for Structural Genomics (JCSG) identified globular proteins as targets for structure determination.
• Standard Nuclear Magnetic Resonance (NMR) experiments are crucial for obtaining polypeptide backbone NMR assignments.

Purpose of the Study:

• To evaluate the efficiency and effectiveness of a standard set of three APSY-NMR experiments for polypeptide backbone NMR assignments in globular proteins.
• To assess the performance of automated data analysis software (UNIO-MATCH-2014) in achieving complete or partial residue assignments.

Main Methods:

• Utilized a standard set of three APSY-NMR experiments for data acquisition.
• Employed automated data analysis with UNIO-MATCH-2014 software for initial assignments.
• Integrated 3D heteronuclear-resolved NOESY spectra for interactive validation and extension of assignments.
• Applied the J-UNIO protocol for NMR structure determination, incorporating automation.

Main Results:

• Achieved complete or partial assignments for over 90% of residues in a representative sample of 30 proteins.
• Completed APSY data acquisition in less than 30 hours for most proteins.
• Obtained high-quality protein structures for all 30 tested proteins.

Conclusions:

• The standard APSY-NMR experiment set, combined with automated analysis, is highly effective for rapid protein structure determination.
• Automated NMR assignment procedures significantly enhance the efficiency of obtaining conformational constraints and determining protein structures.
• The J-UNIO protocol facilitates efficient and automated NMR structure determination, yielding high-quality results.