Protein 3° structure symmetry is a defining feature of nearly one-third of protein folds and is generally thought to result from a combination of gene duplication, fusion, and truncation events. Such events represent major replication errors, involving substantial alteration of protein 3° structure and causing regions of exact repeating 1° structure, both of which are generally considered deleterious to protein folding. Thus, the prevalence of symmetric protein folds is counterintuitive and suggests a specific, yet unexplained, robustness. Using a designed β-trefoil protein, we show that purely symmetric 1° structure enables utilization of alternative definitions of the critical folding nucleus in response to gross structural rearrangement. Thus, major replication errors producing 1° structure symmetry can conserve foldability. The results provide an explanation for the prevalence of symmetric protein folds and highlight a critical role for 1° structure symmetry in protein evolution.

Abstract:The halophile environment has a number of compelling aspects with regard to the origin of structured polypeptides (i.e., proteogenesis) and, instead of a curious niche that living systems adapted into, the halophile environment is emerging as a candidate "cradle" for proteogenesis. In this viewpoint, a subsequent halophile-to-mesophile transition was a key step in early evolution. Several lines of evidence indicate that aromatic amino acids were a late addition to the codon table and not part of the original "prebiotic" set comprising the earliest polypeptides. We test the hypothesis that the availability of aromatic amino acids could facilitate a halophile-to-mesophile transition by hydrophobic core-packing enhancement. The effects of aromatic amino acid substitutions were evaluated in the core of a "primitive" designed protein enriched for the 10 prebiotic amino acids (A,D,E,G,I,L,P,S,T,V)-having an exclusively prebiotic core and requiring halophilic conditions for folding. The results indicate that a single aromatic amino acid substitution is capable of eliminating the requirement of halophile conditions for folding of a "primitive" polypeptide. Thus, the availability of aromatic amino acids could have facilitated a critical halophile-tomesophile protein folding adaptation-identifying a selective advantage for the incorporation of aromatic amino acids into the codon table.

Many protein architectures exhibit evidence of internal rotational symmetry postulated to be the result of gene duplication/fusion events involving a primordial polypeptide motif. A common feature of such structures is a domain-swapped arrangement at the interface of the N-and C-termini motifs and postulated to provide cooperative interactions that promote folding and stability. De novo designed symmetric protein architectures have demonstrated an ability to accommodate circular permutation of the N-and C-termini in the overall architecture; however, the folding requirement of the primordial motif is poorly understood, and tolerance to circular permutation is essentially unknown. The β-trefoil protein fold is a threefold-symmetric architecture where the repeating~42-mer "trefoil-fold" motif assembles via a domainswapped arrangement. The trefoil-fold structure in isolation exposes considerable hydrophobic area that is otherwise buried in the intact β-trefoil trimeric assembly. The trefoil-fold sequence is not predicted to adopt the trefoil-fold architecture in ab initio folding studies; rather, the predicted fold is closely related to a compact "blade" motif from the β-propeller architecture. Expression of a trefoil-fold sequence and circular permutants shows that only the wild-type N-terminal motif definition yields an intact β-trefoil trimeric assembly, while permutants yield monomers. The results elucidate the folding requirements of the primordial trefoil-fold motif, and also suggest that this motif may sample a compact conformation that limits hydrophobic residue exposure, contains key trefoil-fold structural features, but is more structurally homologous to a β-propeller blade motif. K E Y W O R D Sdomain swapping, folding pathway, protein evolution, protein symmetry

An efficient protein-folding pathway leading to target structure, and the avoidance of aggregation, is essential to protein evolution and de novo design; however, design details to achieve efficient folding and avoid aggregation are poorly understood. We report characterization of the thermally-induced aggregate of fibroblast growth factor-1 (FGF-1), a small globular protein, by solid-state NMR. NMR spectra are consistent with residual structure in the aggregate and provide evidence of a structured region that corresponds to the region of the folding nucleus. NMR data on aggregated FGF-1 also indicate the presence of unstructured regions that exhibit hydration-dependent dynamics and suggest that unstructured regions of aggregated FGF-1 lie outside the folding nucleus. Since it is known that regions outside the folding nucleus fold late in the folding pathway, we postulate that these regions unfold early in the unfolding pathway and that the partially folded state is more prone to intermolecular aggregation. This interpretation is further supported by comparison with a designed protein that shares the same FGF-1 folding nucleus sequence, but has different 1° structure outside the folding nucleus, and does not thermally aggregate. The results suggest that design of an efficient folding nucleus, and the avoidance of aggregation in the folding pathway, are potentially separable design criteria - the latter of which could principally focus upon the physicochemical properties of 1° structure outside the folding nucleus.

Gene duplication and fusion events in protein evolution are postulated to be responsible for the common protein folds exhibiting internal rotational symmetry. Such evolutionary processes can also potentially yield regions of repetitive primary structure. Repetitive primary structure offers the potential for alternative definitions of critical regions, such as the folding nucleus (FN). In principle, more than one instance of the FN potentially enables an alternative folding pathway in the face of a subsequent deleterious mutation. We describe the targeted mutation of the carboxyl‐terminal region of the (internally located) FN of the de novo designed purely‐symmetric β‐trefoil protein Symfoil‐4P. This mutation involves wholesale replacement of a repeating trefoil‐fold motif with a “blade” motif from a β‐propeller protein, and postulated to trap that region of the Symfoil‐4P FN in a nonproductive folding intermediate. The resulting protein (termed “Bladefoil”) is shown to be cooperatively folding, but as a trimeric oligomer. The results illustrate how symmetric protein architectures have potentially diverse folding alternatives available to them, including oligomerization, when preferred pathways are perturbed.

The beta‐trefoil protein architecture is characterized by three repeating “trefoil” motifs related by rotational symmetry and postulated to have evolved via gene duplication and fusion events. Despite this apparent structural symmetry, the primary and secondary structural elements typically exhibit pronounced asymmetric features. A survey of this family of proteins has revealed that among the most conserved symmetric structural elements is a ubiquitous buried solvent which participates in a bridging H‐bond with three different beta‐strands in each of the trefoil motifs. A computational analysis reported that these waters are likely associated with a substantial enthalpic contribution to overall stability. In this report, a Pro mutation is used to disrupt one of the water H‐bond interactions to a main chain amide, and the effects upon stability and folding kinetics are determined. Combined with Ala mutations, the separate effects upon side chain truncation and H‐bond deletion are analyzed in terms of stability and folding kinetics. The results show that these buried waters act to assemble a central folding nucleus, and are responsible for ~20% of the overall favorable enthalpy of folding.

Human fibroblast growth factor-1 (FGF-1) has broad therapeutic potential in regenerative medicine but has undesirable biophysical properties of low thermostability and 3 buried cysteine (Cys) residues (at positions 16, 83, and 117) that interact to promote irreversible protein unfolding under oxidizing conditions. Mutational substitution of such Cys residues eliminates reactive buried thiols but cannot be accomplished simultaneously at all 3 positions without also introducing further substantial instability. The mutational introduction of a novel Cys residue (Ala66Cys) that forms a stabilizing disulfide bond (i.e., cystine) with one of the extant Cys residues (Cys83) effectively eliminates one Cys while increasing overall stability. This increase in stability offsets the associated instability of remaining Cys substitution mutations and permits production of a Cys-free form of FGF-1 (Cys16Ser/Ala66Cys/Cys117Ala) with only minor overall instability. The addition of a further stabilizing mutation (Pro134Ala) creates a Cys-free FGF-1 mutant with essentially wild-type biophysical properties. The elimination of buried free thiols in FGF-1 can substantially increase the protein half-life in cell culture. Here, we show that the effective cell survival/mitogenic functional activity of a fully Cys-free form is also substantially increased and is equivalent to wild-type FGF-1 formulated in the presence of heparin sulfate as a stabilizing agent. The results identify this Cys-free FGF-1 mutant as an advantageous “second generation” form of FGF-1 for therapeutic application.

Successful de novo protein design ideally targets specific folding kinetics, stability thermodynamics, and biochemical functionality, and the simultaneous achievement of all these criteria in a single step design is challenging. Protein design is potentially simplified by separating the problem into two steps: (a) an initial design of a protein “scaffold” having appropriate folding kinetics and stability thermodynamics, followed by (b) appropriate functional mutation—possibly involving insertion of a peptide functional “cassette.” This stepwise approach can also separate the orthogonal effects of the “stability/function” and “foldability/function” tradeoffs commonly observed in protein design. If the scaffold is a protein architecture having an exact rotational symmetry, then there is the potential for redundant folding nuclei and multiple equivalent sites of functionalization; thereby enabling broader functional adaptation. We describe such a “scaffold” and functional “cassette” design strategy applied to a β‐trefoil threefold symmetric architecture and a heparin ligand functionality. The results support the availability of redundant folding nuclei within this symmetric architecture, and also identify a minimal peptide cassette conferring heparin affinity. The results also identify an energy barrier of destabilization that switches the protein folding pathway from monomeric to trimeric, thereby identifying another potential advantage of symmetric protein architecture in de novo design.