A General Strategy to Access Structural Information at Atomic Resolution in Polyglutamine Homorepeats

Area of Science:

• Biochemistry and Structural Biology
• Molecular Medicine
• Protein Science

Background:

• Homorepeat (HR) proteins play crucial roles in biological processes and disease.
• The homotypic nature of HR proteins has hindered high-resolution structural characterization.
• Understanding HR protein structure is vital for deciphering pathologies like polyglutamine diseases.

Purpose of the Study:

• To develop a strategy for site-specific isotopic labeling of glutamines within HR proteins.
• To enable high-resolution structural and dynamic investigations of HR proteins, including huntingtin.
• To elucidate structural changes associated with expanded polyglutamine (poly-Q) tracts in disease.

Main Methods:

• Combined nonsense suppression and cell-free expression for site-specific glutamine labeling.
• Utilized Nuclear Magnetic Resonance (NMR) spectroscopy for structural analysis.
• Applied the method to huntingtin exon1 with a 16-residue poly-Q tract and a pathological 46-glutamine variant.

Main Results:

• Successfully achieved isotopic labeling of individual glutamines in HR proteins.
• NMR investigation revealed an N-terminal α-helix in huntingtin exon1 (16Q) at neutral pH, which diminishes along the poly-Q tract.
• Demonstrated the strategy's applicability to pathological huntingtin variants with expanded poly-Q repeats.

Conclusions:

• The developed methodology enables high-resolution structural characterization of HR proteins.
• This approach provides insights into structural and dynamic perturbations in poly-Q-related diseases.
• The strategy is extendable to other amino acids and proteins with low-complexity regions (LCRs).