A reduced dimensionality NMR pulse sequence and an efficient protocol for unambiguous assignment in intrinsically...

Area of Science:

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

Background:

• Intrinsically disordered proteins (IDPs) present significant challenges for resonance assignment in NMR due to poor chemical shift dispersion.
• Reduced dimensionality (RD) NMR experiments offer partial solutions by co-evolving multiple nuclei simultaneously.
• Optimizing nuclei combinations in RD experiments is crucial for enhancing spectral dispersion and information content.

Purpose of the Study:

• To present a novel RD NMR experiment, (4,3)D-hNCOCAnH, for improved resonance assignment in IDPs.
• To demonstrate the utility of combining Carbonyl (CO) and Carbon-alpha (CA) chemical shifts for enhanced spectral dispersion.
• To provide a single-experiment method for obtaining chemical shift information for four backbone atoms (HN, N, CA, CO) per residue.

Main Methods:

• Development and application of the (4,3)D-hNCOCAnH RD NMR experiment.
• Utilizing co-evolution of CO and CA chemical shifts along a spectral axis.
• Employing unidirectional sequential (i → i - 1) amide (1)H correlations.
• Demonstration using the intrinsically disordered α-synuclein protein (140 aa).

Main Results:

• The (4,3)D-hNCOCAnH experiment significantly improves spectral dispersion for IDPs.
• It enables the collection of HN, N, CA, and CO chemical shift information from a single experiment.
• The experiment facilitates straightforward assignment of most protein backbone nuclei through sequential correlations.
• Potential ambiguities in amide proton chemical shifts can be resolved using complementary experiments like HNN.

Conclusions:

• The (4,3)D-hNCOCAnH experiment is a valuable tool for tackling resonance assignment challenges in intrinsically disordered proteins.
• This method enhances spectral resolution and provides comprehensive backbone atom information, simplifying the assignment process.
• The demonstrated application on α-synuclein highlights its practical utility in structural biology research.