Functionalization of a symmetric protein scaffold: Redundant folding nuclei and alternative oligomeric folding pathways.

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 ß-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.

The ubiquitous buried water in the beta-trefoil architecture contributes to the folding nucleus and ~20% of the folding enthalpy.

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.

Oligomerization of a symmetric ß-trefoil protein in response to folding nucleus perturbation.

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 ß-trefoil protein Symfoil-4P. This mutation involves wholesale replacement of a repeating trefoil-fold motif with a "blade" motif from a ß-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.

Ab initio folding of a trefoil-fold motif reveals structural similarity with a ß-propeller blade motif.

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 ß-trefoil protein fold is a threefold-symmetric architecture where the repeating ~42-mer "trefoil-fold" motif assembles via a domain-swapped arrangement. The trefoil-fold structure in isolation exposes considerable hydrophobic area that is otherwise buried in the intact ß-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 ß-propeller architecture. Expression of a trefoil-fold sequence and circular permutants shows that only the wild-type N-terminal motif definition yields an intact ß-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 ß-propeller blade motif.

Investigating the dynamics and polyanion binding sites of fibroblast growth factor-1 using hydrogen-deuterium exchange mass spectrometry.

In this study, we examined the local dynamics of acidic fibroblast growth factor (FGF-1) as well as the binding sites of various polyanions including poly-sulfates (heparin and low MW heparin) and poly-phosphates (phytic acid and ATP) using hydrogen-deuterium exchange mass spectrometry (HX-MS). For local dynamics, results are analyzed at the peptide level as well as in terms of buried amides employing crystallographic B-factors and compared with a residue level heat map generated from HX-MS results. Results show that strand 4 and 5 and the turn between them to be the most flexible regions as was previously seen by NMR. On the other hand, the C-terminal strands 8, 9, and 10 appear to be more rigid which is also consistent with crystallographic B-factors as well as local dynamics studies conducted by NMR. Crystal structures of FGF-1 in complex with heparin have shown that heparin binds to N-terminal Asn18 and to C-terminal Lys105, Tryp107, Lys112, Lys113, Arg119, Pro121, Arg122, Gln127, and Lys128 indicating electrostatic forces as dominant interactions. Heparin binding as determined by HX-MS is consistent with crystallography data. Previous studies have also shown that other polyanions including low MW heparin, phytic acid and ATP dramatically increase the thermal stability of FGF-1. Using HX-MS, we find other poly anions tested bind in a similar manner to heparin, primarily targeting the turns in the lysine rich C-terminal region of FGF-1 along with two distinct N-terminal regions that contains lysines and arginines/histidines. This confirms the interactions between FGF-1 and polyanions are primary directed by electrostatics.

Fine-Sampled Photographic Quantitation of Dermal Wound Healing Senescence in Aged BALB/cByJ Mice and Therapeutic Intervention with Fibroblast Growth Factor-1.

Objective: To determine quantitative parameters of dermal wound healing senescence in aged BALB/cByJ mice (an important animal model of aging) and to evaluate the potential for therapeutic intervention by fibroblast growth factor-1 (FGF-1). Approach: Utilize a novel noninvasive fine-sampled photographic methodology to quantify wound healing parameters for healing phases from wounding through to wound closure. Results: Parameters associated with key healing phases were quantified and compared between nonaged and aged cohorts of both genders. The results identify a sexual dimorphism in dermal wound healing, with nonaged females exhibiting a greater overall healing efficiency than males. This enhanced healing in females, however, senesces with age such that healing parameters for aged males and females are statistically indistinguishable. Topical application of FGF-1 was identified as an effective therapeutic intervention to treat dermal healing senescence in aged females. Innovation: The FGF intervention is being analyzed using a new recently published model. This approach significantly increases the amount of preclinical animal data obtainable in wound healing studies, minimizes cohort number compared with (lethal) histological studies, and permits a direct statistical comparison between different healing studies. Conclusion: Quantitative parameters of dermal wound healing, obtained from noninvasive fine-sampled photographic data, identify topical FGF-1 as an effective therapeutic to treat the senescence of dermal healing present in aged female BALB/cByJ mice.

Folding nucleus structure persists in thermally-aggregated FGF-1.

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.

An S116R Phosphorylation Site Mutation in Human Fibroblast Growth Factor-1 Differentially Affects Mitogenic and Glucose-Lowering Activities.

Fibroblast growth factor-1 (FGF-1), a potent human mitogen and insulin sensitizer, signals through both tyrosine kinase receptor-mediated autocrine/paracrine pathways as well as a nuclear intracrine pathway. Phosphorylation of FGF-1 at serine 116 (S116) has been proposed to regulate intracrine signaling. Position S116 is located within a ∼17 amino acid C-terminal loop that contains a rich set of functional determinants including heparin∖heparan sulfate affinity, thiol reactivity, nuclear localization, pharmacokinetics, functional half-life, nuclear ligand affinity, stability, and structural dynamics. Mutational targeting of specific functionality in this region without perturbing other functional determinants is a design challenge. S116R is a non-phosphorylatable variant present in bovine FGF-1 and other members of the human FGF family. We show that the S116R mutation in human FGF-1 is accommodated with no perturbation of biophysical or structural properties, and is therefore an attractive mutation with which to elucidate the functional role of phosphorylation. Characterization of S116R shows reduction in NIH 3T3 fibroblast mitogenic stimulation, increase in fibroblast growth factor receptor-1c activation, and prolonged duration of glucose lowering in ob/ob hyperglycemic mice. A novel FGF-1/fibroblast growth factor receptor-1c dimerization interaction combined with non-phosphorylatable intracrine signaling is hypothesized to be responsible for these observed functional effects.

Engineering a Cysteine-Free Form of Human Fibroblast Growth Factor-1 for "Second Generation" Therapeutic Application.

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.

A single aromatic core mutation converts a designed "primitive" protein from halophile to mesophile folding.

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-to-mesophile protein folding adaptation-identifying a selective advantage for the incorporation of aromatic amino acids into the codon table.

Alternative folding nuclei definitions facilitate the evolution of a symmetric protein fold from a smaller peptide motif.

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 ß-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.