Abstract
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.
Highlights
The α-helical coiled-coil motif, one of the most abundant structural protein motifs found in nature, represents a convenient and widely used system, as it combines structural simplicity, remarkable functional versatility, and structural plasticity reflected in a large variety of topologies and folding states [1,2,3]
The amino acid sequences of α-helices in coiled-coil helical bundles are characterized by a pattern of seven-residue quasi-repeats (“heptads”) of the kind (a, b, c, d, e, f, g)n [8], where n is the number of repeats and a–g are the topologically distinct positions of the amino acids in a heptad (Figure 1)
These apolar residues define a hydrophobic stripe which forms the interface between the associating α-helices and the core of the bundle, and pack along it, in a “knobs-into-hole” fashion to minimize their interaction with water molecules [9]
Summary
The occurrence of hydrophobic residues at the a, d positions of the heptad repeats is a hallmark feature of coiled-coils These apolar residues define a hydrophobic stripe which forms the interface between the associating α-helices and the core of the bundle, and pack along it, in a “knobs-into-hole” fashion to minimize their interaction with water molecules [9]. Detailed position-specific amino acid preferences of four-α-helical bundles have been described by Paliakasis and Kokkinidis [8]. These insights into coiled-coil sequence-to-structure relationships rely to a large extent on protein structure determinations at high resolutions. Due to the structure of the amino acids, a polypeptide chain has directionality, meaning that it has two ends which are chemically distinct from one another, affecting folding differently
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