A new strategy for backbone resonance assignment in large proteins using a MQ-HACACO experiment

Area of Science:

• Structural Biology
• Biophysics
• Nuclear Magnetic Resonance Spectroscopy

Background:

• Backbone resonance assignment is crucial for protein structure determination.
• Traditional methods face challenges with larger proteins due to relaxation effects.
• Sensitive TROSY-type experiments offer improved performance for larger biomolecules.

Purpose of the Study:

• To develop a new strategy for backbone resonance assignment in large proteins.
• To leverage favorable relaxation properties of multiple-quantum coherences.
• To enable assignment of (1)H(alpha) resonances, which is challenging in existing methods.

Main Methods:

• Combination of TROSY-HNCA and TROSY-HNCO experiments.
• Introduction of a novel 3D multiple-quantum HACACO experiment.
• Utilizing (13)C' antiphase coherences for signal detection.
• Application to uniformly (15)N,(13)C-labeled and fractionally deuterated trimeric B. subtilis Chorismate Mutase (44 kDa).

Main Results:

• The proposed strategy demonstrates efficient backbone resonance assignment.
• The 3D multiple-quantum HACACO experiment successfully assigns (1)H(alpha) resonances.
• The method is effective at both 20°C and 9°C.
• Successful application to a 44 kDa protein at standard and lower temperatures.

Conclusions:

• The new strategy significantly optimizes protein resonance assignment for larger proteins.
• The method's applicability extends to proteins up to 80 kDa at lower temperatures.
• This approach advances NMR capabilities for studying large protein systems.