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.

Structural symmetry is observed in the majority of fundamental protein folds and gene duplication and fusion evolutionary processes are postulated to be responsible. However, convergent evolution leading to structural symmetry has also been proposed; additionally, there is debate regarding the extent to which exact primary structure symmetry is compatible with efficient protein folding. Issues of symmetry in protein evolution directly impact strategies for de novo protein design as symmetry can substantially simplify the design process. Additionally, when considering gene duplication and fusion in protein evolution, there are two competing models: “emergent architecture” and “conserved architecture”. Recent experimental work has shed light on both the evolutionary process leading to symmetric protein folds as well as the ability of symmetric primary structure to efficiently fold. Such studies largely support a “conserved architecture” evolutionary model, suggesting that complex protein architecture was an early evolutionary achievement involving oligomerization of smaller polypeptides.

The majority of protein architectures exhibit elements of structural symmetry, and “gene duplication and fusion” is the evolutionary mechanism generally hypothesized to be responsible for their emergence from simple peptide motifs. Despite the central importance of the gene duplication and fusion hypothesis, experimental support for a plausible evolutionary pathway for a specific protein architecture has yet to be effectively demonstrated. To address this question, a unique “top-down symmetric deconstruction” strategy was utilized to successfully identify a simple peptide motif capable of recapitulating, via gene duplication and fusion processes, a symmetric protein architecture (the threefold symmetric β-trefoil fold). The folding properties of intermediary forms in this deconstruction agree precisely with a previously proposed “conserved architecture” model for symmetric protein evolution. Furthermore, a route through foldable sequence-space between the simple peptide motif and extant protein fold is demonstrated. These results provide compelling experimental support for a plausible evolutionary pathway of symmetric protein architecture via gene duplication and fusion processes.

Buried free-cysteine (Cys) residues can contribute to an irreversible unfolding pathway that promotes protein aggregation, increases immunogenic potential, and significantly reduces protein functional half-life. Consequently, mutation of buried free-Cys residues can result in significant improvement in the storage, reconstitution, and pharmacokinetic properties of protein-based therapeutics. Mutational design to eliminate buried free-Cys residues typically follows one of two common heuristics: either substitution by Ser (polar and isosteric), or substitution by Ala or Val (hydrophobic); however, a detailed structural and thermodynamic understanding of Cys mutations is lacking. We report a comprehensive structure and stability study of Ala, Ser, Thr, and Val mutations at each of the three buried free-Cys positions (Cys16, Cys83, and Cys117) in fibroblast growth factor-1. Mutation was almost universally destabilizing, indicating a general optimization for the wild-type Cys, including van der Waals and H-bond interactions. Structural response to Cys mutation characteristically involved changes to maintain, or effectively substitute, local H-bond interactions—by either structural collapse to accommodate the smaller oxygen radius of Ser/Thr, or conversely, expansion to enable inclusion of novel H-bonding solvent. Despite the diverse structural effects, the least destabilizing average substitution at each position was Ala, and not isosteric Ser. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:566–576, 2015

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.

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 folded β-trefoil protein was designed by symmetric expansion of a folding nucleus•The β-trefoil protein is more stable, less aggregation prone, than its progenitor•Folding nucleus symmetric expansion (FNSE) is an efficient approach to protein design•FNSE may be applied to any symmetric protein fold (∼one-third of known architectures)Models of symmetric protein evolution typically invoke gene duplication and fusion events, in which repetition of a structural motif generates foldable, stable symmetric protein architecture. Success of such evolutionary processes suggests that the duplicated structural motif must be capable of nucleating protein folding. If correct, symmetric expansion of a folding nucleus sequence derived from an extant symmetric fold may be an elegant and computationally tractable solution to de novo protein design. We report the efficient de novo design of a β-trefoil protein by symmetric expansion of a β-trefoil folding nucleus, previously identified by ɸ-value analysis. The resulting protein, having exact sequence symmetry, exhibits superior folding properties compared to its naturally evolved progenitor—with the potential for redundant folding nuclei. In principle, folding nucleus symmetric expansion can be applied to any given symmetric protein fold (that is, nearly one-third of the known proteome) provided information of the folding nucleus is available.Download high-res image (514KB)Download full-size image

•Evolution of symmetric protein folds involves duplicated regions of 1° structure•Structural rearrangement of a purely symmetric protein does not perturb folding kinetics•Symmetry can maintain foldability despite major structural rearrangements in evolutionProtein 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.Download high-res image (222KB)Download full-size image

Turn secondary structure is essential to the formation of globular protein architecture. Turn structures are, however, much more complex than either α-helix or β-sheet, and the thermodynamics and folding kinetics are poorly understood. Type I β-turns are the most common type of reverse turn, and they exhibit a statistical consensus sequence of Asx-Pro-Asx-Gly (where Asx is Asp or Asn). A comprehensive series of individual and combined Asx mutations has been constructed within three separate type I 3:5 G1 bulge β-turns in human fibroblast growth factor-1, and their effects on structure, stability, and folding have been determined. The results show a fundamental logical OR relationship between the Asx residues in the motif, involving H-bond interactions with main-chain amides within the turn. These interactions can be modulated by additional interactions with residues adjacent to the turn at positions i + 4 and i + 6. The results show that the Asx residues in the turn motif make a substantial contribution to the overall stability of the protein, and the Asx logical OR relationship defines a redundant system that can compensate for deleterious point mutations. The results also show that the stability of the turn is unlikely to be the prime determinant of formation of turn structure in the folding transition state.

The β-trefoil protein human fibroblast growth factor-1 (FGF-1) is made up of a six-stranded antiparallel β-barrel closed off on one end by three β-hairpins, thus exhibiting a 3-fold axis of structural symmetry. The N and C terminus β-strands hydrogen bond to each other and their interaction is postulated from both NMR and X-ray structure data to be important in folding and stability. Specific mutations within the adjacent N and C terminus β-strands of FGF-1 are shown to provide a substantial increase in stability. This increase is largely correlated with an increased folding rate constant, and with a smaller but significant decrease in the unfolding rate constant. A series of stabilizing mutations are subsequently combined and result in a doubling of the ΔG value of unfolding. When taken in the context of previous studies of stabilizing mutations, the results indicate that although FGF-1 is known for generally poor thermal stability, the β-trefoil architecture appears capable of substantial thermal stability. Targeting stabilizing mutations within the N and C terminus β-strand interactions of a β-barrel architecture may be a generally useful approach to increase protein stability. Such stabilized mutations of FGF-1 are shown to exhibit significant increases in effective mitogenic potency, and may prove useful as “second generation” forms of FGF-1 for application in angiogenic therapy.

Protein biopharmaceuticals are an important and growing area of human therapeutics; however, the intrinsic property of proteins to adopt alternative conformations (such as during protein unfolding and aggregation) presents numerous challenges, limiting their effective application as biopharmaceuticals. Using fibroblast growth factor-1 as model system, we describe a cooperative interaction between the intrinsic property of thermostability and the reactivity of buried free-cysteine residues that can substantially modulate protein functional half-life. A mutational strategy that combines elimination of buried free cysteines and secondary mutations that enhance thermostability to achieve a substantial gain in functional half-life is described. Furthermore, the implementation of this design strategy utilizing stabilizing mutations within the core region resulted in a mutant protein that is essentially indistinguishable from wild type as regard protein surface and solvent structure, thus minimizing the immunogenic potential of the mutations. This design strategy should be generally applicable to soluble globular proteins containing buried free-cysteine residues.

The 22 members of the mouse/human fibroblast growth factor (FGF) family of proteins contain a conserved cysteine residue at position 83 (numbering scheme of the 140-residue form of FGF-1). Sequence and structure information suggests that this position is a free cysteine in 16 members and participates as a half-cystine in at least 3 (and perhaps as many as 6) other members. While a structural role as a half-cystine provides a stability basis for possible selective pressure, it is less clear why this residue is conserved as a free cysteine (although free buried thiols can limit protein functional half-life). To probe the structural role of the free cysteine at position 83 in FGF-1, we constructed Ala, Ser, Thr, Val, and Ile mutations and determined their effects on structure and stability. These results show that position 83 in FGF-1 is thermodynamically optimized to accept a free cysteine. A second cysteine mutation was introduced into wild-type FGF-1 at adjacent position Ala66, which is known to participate as a half-cystine with position 83 in FGF-8, FGF-19, and FGF-23. Results show that, unlike position 83, a free cysteine at position 66 destabilizes FGF-1; however, upon oxidation, a near-optimal disulfide bond is formed between Cys66 and Cys83, resulting in ∼ 14 kJ/mol of increased thermostability. Thus, while the conserved free cysteine at position 83 in the majority of the FGF proteins may have a principal role in limiting functional half-life, evidence suggests that it is a vestigial half-cystine.

Fibroblast growth factor-1, a member of the 3-fold symmetric β-trefoil fold, was subjected to a series of symmetric constraint mutations in a process termed “top-down symmetric deconstruction.” The mutations enforced a cumulative exact 3-fold symmetry upon symmetrically equivalent positions within the protein and were combined with a stability screen. This process culminated in a β-trefoil protein with exact 3-fold primary-structure symmetry that exhibited excellent folding and stability properties. Subsequent fragmentation of the repeating primary-structure motif yielded a 42-residue polypeptide capable of spontaneous assembly as a homotrimer, producing a thermostable β-trefoil architecture. The results show that despite pronounced reduction in sequence complexity, pure symmetry in the design of a foldable, thermostable β-trefoil fold is possible. The top-down symmetric deconstruction approach provides a novel alternative means to successfully identify a useful polypeptide “building block” for subsequent “bottom-up” de novo design of target protein architecture.Download high-res image (167KB)Download full-size imageResearch Highlights► Sequential symmetric constraint mutations applied to FGF-1 produced a 3-fold symmetric primary structure. ► The 42-residue repeating peptide motif spontaneously assembles as a homotrimer to produce a β-trefoil fold. ► This novel approach provides an alternative strategy to the design of peptide “building blocks” for the de novo protein design of symmetric protein architecture.

∆1-Tetrahydrocannabinolic acid (THCA) synthase catalyzes the oxidative cyclization of cannabigerolic acid (CBGA) into THCA, the precursor of the primary psychoactive agent ∆1-tetrahydrocannabinol in Cannabis sativa. The enzyme was overproduced in insect cells, purified, and crystallized in order to investigate the structure–function relationship of THCA synthase, and the tertiary structure was determined to 2.75 Å resolution by X-ray crystallography (Rcryst = 19.9%). The THCA synthase enzyme is a member of the p-cresol methyl-hydroxylase superfamily, and the tertiary structure is divided into two domains (domains I and II), with a flavin adenine dinucleotide coenzyme positioned between each domain and covalently bound to His114 and Cys176 (located in domain I). The catalysis of THCA synthesis involves a hydride transfer from C3 of CBGA to N5 of flavin adenine dinucleotide and the deprotonation of O6′ of CBGA. The ionized residues in the active site of THCA synthase were investigated by mutational analysis and X-ray structure. Mutational analysis indicates that the reaction does not involve the carboxyl group of Glu442 that was identified as the catalytic base in the related berberine bridge enzyme but instead involves the hydroxyl group of Tyr484. Mutations at the active‐site residues His292 and Tyr417 resulted in a decrease in, but not elimination of, the enzymatic activity of THCA synthase, suggesting a key role for these residues in substrate binding and not direct catalysis.Download high-res image (307KB)Download full-size imageHighlights► THCA synthase catalyzes the oxidative cyclization of CBGA into THCA. ► The structure-function relationship of THCA synthase was investigated. ► X-ray crystal structure of THCA synthase was determined to 2.75 Å resolution. ► Specific residues of THCA synthase were identified by mutational analysis.