Probing Protein Folding with Sequence-Reversed α-Helical Bundles

Area of Science:

• Biochemistry
• Structural Biology
• Protein Science

Background:

• Recurrent protein folding motifs often involve α-helices supercoiling to form bundles.
• Four-α-helical bundles are characterized by specific amino acid residue patterns known as heptad repeats.
• The role of polypeptide chain directionality in the folding and oligomerization of these bundles is not fully understood.

Purpose of the Study:

• To investigate the impact of polypeptide chain directionality on sequence-structure relationships in four-α-helical bundles.
• To analyze how reversing amino acid sequences affects protein folding, oligomerization, and structural properties.

Main Methods:

• Reversed amino acid sequences of two well-characterized four-α-helical bundle proteins to create retro-proteins.
• Studied folding, oligomerization, and structural properties using Circular Dichroism Spectroscopy (CD), Size Exclusion Chromatography with Multi-Angle Laser Light Scattering (SEC-MALS), and Small Angle X-ray Scattering (SAXS).

Main Results:

• Sequence reversal affected the α-helical character of the parent proteins to varying degrees.
• Folding states, oligomerization propensities, structural stabilities, and shapes of retro-proteins depended significantly on heptad repeat pattern characteristics.
• Highest similarities between parent and retro-proteins were observed with uninterrupted heptad patterns.

Conclusions:

• Polypeptide chain directionality plays a crucial role in the folding and structural integrity of four-α-helical bundles.
• Heptad repeat patterns are key determinants of structural stability and oligomerization in these bundles, even upon sequence reversal.
• Uninterrupted heptad patterns are critical for maintaining structural similarity between native and reversed protein sequences.