Simplified protein design biased for prebiotic amino acids yields a foldable, halophilic protein

Abstract

A compendium of different types of abiotic chemical syntheses identifies a consensus set of 10 "prebiotic" α-amino acids. Before the emergence of biosynthetic pathways, this set is the most plausible resource for protein formation (i.e., proteogenesis) within the overall process of abiogenesis. An essential unsolved question regarding this prebiotic set is whether it defines a "foldable set"--that is, does it contain sufficient chemical information to permit cooperatively folding polypeptides? If so, what (if any) characteristic properties might such polypeptides exhibit? To investigate these questions, two "primitive" versions of an extant protein fold (the β-trefoil) were produced by top-down symmetric deconstruction, resulting in a reduced alphabet size of 12 or 13 amino acids and a percentage of prebiotic amino acids approaching 80%. These proteins show a substantial acidification of pI and require high salt concentrations for cooperative folding. The results suggest that the prebiotic amino acids do comprise a foldable set within the halophile environment.

Fig. 1.

Amino acid sequence of FGF-1, Symfoil-4P, PV1, and PV2 mutants (single letter code). The sequences are aligned by the threefold axis of rotational symmetry internal to the β-trefoil fold (i.e., each line is one trefoil-fold repeat element within the overall β-trefoil structure). The numbering of Symfoil-4P, PV1, and PV2 amino acids is based upon corresponding positions in FGF-1. The shaded positions identify amino acids belonging to the prebiotic set.

Fig. 2.

Amino acid composition of FGF-1, Symfoil-4P, PV1, and PV2 mutants. Also shown are the alphabet size, charge bias, pI, number of aromatic residues, and % prebiotic amino acids for each protein. The shaded positions identify amino acids belonging to the prebiotic set.

Fig. 3.

DSC endotherms collected in the presence of low (0.1 M) and high (2.0 M) NaCl. All proteins exhibit increased thermal stability under the high-salt condition. However, although the FGF-1 protein is insoluble upon thermal denaturation under both low- and high-salt conditions, the Symfoil-4P, PV1, and PV2 proteins remain soluble. Under low-salt conditions, the PV2 protein, although soluble, is partially unfolded; however, under high-salt conditions the PV2 protein is essentially fully folded. Thus, PV2 exhibits characteristics of an obligate halophile.

Fig. 4.

Surface electrostatic charge distribution of FGF-1 and PV2 proteins. Beneath each surface representation is an associated ribbon diagram in the same orientation. The left-most image of each pair is a side view; the right-most image is rotated 90° about the horizontal axis to provide a bottom view of the overall β-barrel structure. Positive charge density is indicated by blue, and negative by red. The (a)symmetric features of each protein can be appreciated in the right-most (bottom) view in each case. The acidic nature of the prebiotic set of amino acids is evident with the enrichment of such residues in the PV2 protein.