Probing Protein Folding with Sequence-Reversed α-Helical Bundles

• Published in: International journal of molecular sciences Vol. 22; no. 4; p. 1955
• Main Authors: Kefala, Aikaterini, Amprazi, Maria, Mylonas, Efstratios, Kotsifaki, Dina, Providaki, Mary, Pozidis, Charalambos, Fotiadou, Melina, Kokkinidis, Michael
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI 16.02.2021
• Subjects: Amino Acid Motifs; Amino Acid Sequence; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Chromatography, Gel; Circular Dichroism; Escherichia coli - genetics; Escherichia coli - metabolism; Models, Molecular; Peptides; Protein Conformation, alpha-Helical; Protein Folding; RNA-Binding Proteins - chemistry; RNA-Binding Proteins - genetics; Scattering, Small Angle; X-Ray Diffraction; coiled-coil proteins; α-helix; polypeptide chain directionality; circular dichroism (CD); protein folding; 4-α-helical bundle; RM6 protein; rop protein; multiangle laser light scattering (MALS); small angle X-ray scattering (SAXS)
• ISSN: 1422-0067; 1422-0067
• DOI: 10.3390/ijms22041955

Recurrent protein folding motifs include various types of helical bundles formed by α-helices that supercoil around each other. While specific patterns of amino acid residues (heptad repeats) characterize the highly versatile folding motif of four-α-helical bundles, the significance of the polypeptide chain directionality is not sufficiently understood, although it determines sequence patterns, helical dipoles, and other parameters for the folding and oligomerization processes of bundles. To investigate directionality aspects in sequence-structure relationships, we reversed the amino acid sequences of two well-characterized, highly regular four-α-helical bundle proteins and studied the folding, oligomerization, and structural properties of the retro-proteins, using Circular Dichroism Spectroscopy (CD), Size Exclusion Chromatography combined with Multi-Angle Laser Light Scattering (SEC-MALS), and Small Angle X-ray Scattering (SAXS). The comparison of the parent proteins with their retro-counterparts reveals that while the α-helical character of the parents is affected to varying degrees by sequence reversal, the folding states, oligomerization propensities, structural stabilities, and shapes of the new molecules strongly depend on the characteristics of the heptad repeat patterns. The highest similarities between parent and retro-proteins are associated with the presence of uninterrupted heptad patterns in helical bundles sequences.