Effects of chirality and side chain length in Cα,α-dialkylated residues on ß-hairpin peptide folded structure and stability.

Strategic incorporation of achiral Cα,α-dialkylated amino acids with bulky substituents into peptides can be used to promote extended strand conformations and inhibit protein-protein interactions associated with amyloid formation. In this work, we evaluate the thermodynamic impact of chiral Cα,α monomers on folding preferences in such systems through introduction of a series of Cα-methylated and Cα-ethylated residues into a ß-hairpin host sequence. Depending on stereochemical configuration of the artificial monomer and potential for additional hydrophobic packing, a Cα-ethyl-Cα-propyl glycine residue can provide similar or enhanced folded stability relative to an achiral Cα,α-diethyl analogue.

Hydrolysis of a Ni-Schiff-Base Complex Using Conditions Suitable for Retention of Acid-labile Protecting Groups.

Unnatural amino acids, amino acids containing side-chain functionalities not commonly seen in nature, are increasingly found in synthetic peptide sequences. Synthesis of some unnatural amino acids often includes the use of a precursor consisting of a Schiff-base stabilized by a nickel cation. Unnatural side-chains can be installed on an amino acid backbone found in this Schiff-base complex. The resulting unnatural amino acid can then be isolated from this complex using hydrolysis of the Schiff-base, typically by employing reflux in strongly acidic solution. These highly acidic conditions may remove acid-labile side-chain protecting groups necessary for the unnatural amino acids to be used in microwave-assisted solid-phase peptide synthesis. In this work, we present an efficient hydrolysis and subsequent Fmoc protection of an amino acid isolated from a Ni-Schiff base complex. Hydrolysis conditions presented in this work are suitable for retention of acid-labile side-chain protecting groups and may be adaptable to a variety of unnatural amino acid substrates.

Peptide-functionalized semiconductor surfaces: strong surface electronic effects from minor alterations to backbone composition.

The use of non-canonical amino acids is a powerful way to control protein structure. Here, we show that subtle changes to backbone composition affect the ability of a dipeptide to modify solid surface electronic properties. The extreme sensitivity of the interactions to the peptide structure suggests potential applications in improving the performance of electronic devices.

Backbone Engineering within a Latent ß-Hairpin Structure to Design Inhibitors of Polyglutamine Amyloid Formation.

Candidates for the toxic molecular species in the expanded polyglutamine (polyQ) repeat diseases range from various types of aggregates to "misfolded" monomers. One way to vet these candidates is to develop mutants that restrict conformational landscapes. Previously, we inserted two self-complementary ß-hairpin enhancing motifs into a short polyQ sequence to generate a mutant, here called "ßHP," that exhibits greatly improved amyloid nucleation without measurably enhancing ß-structure in the monomer ensemble. We extend these studies here by introducing single-backbone H-bond impairing modifications αN-methyl Gln or l-Pro at key positions within ßHP. Modifications predicted to allow formation of a fully H-bonded ß-hairpin at the fibril edge while interfering with H-bonding to the next incoming monomer exhibit poor amyloid formation and act as potent inhibitors in trans of simple polyQ peptide aggregation. In contrast, a modification that disrupts intra-ß-hairpin H-bonding within ßHP, while also aggregating poorly, is ineffective at inhibiting amyloid formation in trans. The inhibitors constitute a dynamic version of the edge-protection negative design strategy used in protein evolution to limit unwanted protein aggregation. Our data support a model in which polyQ peptides containing strong ß-hairpin encouraging motifs only rarely form ß-hairpin conformations in the monomer ensemble, but nonetheless take on such conformations at key steps during amyloid formation. The results provide insights into polyQ solution structure and fibril formation while also suggesting an approach to the design of inhibitors of polyQ amyloid growth that focuses on conformational requirements for fibril and nucleus elongation.

Thermodynamic and Structural Impact of α,α-Dialkylated Residue Incorporation in a ß-Hairpin Peptide.

Peptides containing α,α-dialkylated α-amino acids, owing to their ability to disrupt aggregation of ß-amyloid proteins, have therapeutic potential in the treatment of neurodegenerative diseases. Thermodynamic and structural analyses are reported for a series of ß-hairpin peptides containing α,α-dialkylated α-amino acids with varying side-chain lengths. The results of these experiments show that α,α-dialkylated α-amino acids with side-chain lengths longer than one carbon unit are tolerated in a ß-hairpin, although at a moderate cost to folded stability.

Comparison of design strategies for α-helix backbone modification in a protein tertiary fold.

We report here the comparison of five classes of unnatural amino acid building blocks for their ability to be accommodated into an α-helix in a protein tertiary fold context. High-resolution structural characterization and analysis of folding thermodynamics yield new insights into the relationship between backbone composition and folding energetics in α-helix mimetics and suggest refined design rules for engineering the backbones of natural sequences.

Comparison of backbone modification in protein ß-sheets by α→γ residue replacement and α-residue methylation.

The mimicry of protein tertiary structure by oligomers with unnatural backbones is a significant contemporary research challenge. Among common elements of secondary structure found in natural proteins, sheets have proven the most difficult to address. Here, we report the systematic comparison of different strategies for peptide backbone modification in ß-sheets with the goal of identifying the best method for replacing a multi-stranded sheet in a protein tertiary fold. The most effective sheet modifications examined led to native-like tertiary folding behavior with a thermodynamic folded stability comparable to the prototype protein on which the modified backbones are based.

Protein-like tertiary folding behavior from heterogeneous backbones.

Because proteins play vital roles in life, much effort has been invested in their mimicry by synthetic agents. One approach is to design unnatural backbone oligomers ("foldamers") that fold like natural peptides. Despite success in secondary structure mimicry by such species, protein-like tertiary folds remain elusive. A fundamental challenge underlying this task is the design of a sequence of side chains that will specify a complex tertiary folding pattern on an unnatural backbone. We report here a sequence-based approach to convert a natural protein with a compact tertiary fold to an analogue with a backbone composed of ~20% unnatural building blocks but folding behavior similar to that of the parent protein.

Introduction of cyclically constrained γ-residues stabilizes an α-peptide hairpin in aqueous solution.

The synthesis and structural characterization of hybrid α/γ-peptides resulting from a 1:1 α→γ residue substitution at cross-strand positions in a hairpin-forming α-peptide sequence are described. Cyclically constrained γ-residues based on 1,3-substituted cyclohexane or benzene scaffolds support a native-like hairpin fold in aqueous solution, and the unnatural residues stabilize the folded state by ∼0.2 kcal/mol per α→γ substitution.

Design strategies for the sequence-based mimicry of side-chain display in protein ß-sheets by α/ß-peptides.

The sophistication of folding patterns and functions displayed by unnatural-backbone oligomers has increased tremendously in recent years. Design strategies for the mimicry of tertiary structures seem within reach; however, a general method for the mimicry of sheet segments in the context of a folded protein is an unmet need preventing realization of this goal. Previous work has shown that 1→1 α→ß-residue substitutions at cross-strand positions in a hairpin-forming α-peptide sequence can generate an α/ß-peptide analogue that folds in aqueous conditions but with a change in side-chain display relative to the natural sequence; this change would prevent application of single ß-residue substitutions in a larger protein. Here, we evaluate four different substitution strategies based on replacement of αα dipeptide segments for the ability to retain both sheet folding encoded by a parent α-peptide sequence as well as nativelike side-chain display in the vicinity of the ß-residue insertion point. High-resolution structure determination and thermodynamic analysis of folding by multidimensional NMR suggest that three of the four designs examined are applicable to larger proteins.

Hairpin folding behavior of mixed α/ß-peptides in aqueous solution.

The invention of new strategies for the design of protein-mimetic oligomers that manifest the folding encoded in natural amino acid sequences is a significant challenge. In contrast to the α-helix, mimicry of protein ß-sheets is less understood. We report here the aqueous folding behavior of a prototype α-peptide hairpin model sequence varied at cross-strand positions by incorporation of 16 different ß-amino acid monomers. Our results provide a folding propensity scale for ß-residues in a protein ß-sheet context as well as high-resolution structures of several mixed-backbone α/ß-peptide hairpins in water.