New charge-bearing amino acid residues that promote ß-sheet secondary structure.

Proteinogenic amino acid residues that promote ß-sheet secondary structure are hydrophobic (e.g., Ile or Val) or only moderately polar (e.g., Thr). The design of peptides intended to display ß-sheet secondary structure in water typically requires one set of residues to ensure conformational stability and an orthogonal set, with charged side chains, to ensure aqueous solubility and discourage self-association. Here we describe new amino acids that manifest substantial ß-sheet propensity, by virtue of ß-branching, and also bear an ionizable group in the side chain.

A γ-amino acid that favors 12/10-helical secondary structure in α/γ-peptides.

H-bonded helices in conventional peptides (containing exclusively homochiral α-amino acid residues) feature a uniform H-bonding directionality, N-terminal side C═O to C-terminal side NH. In contrast, heterochiral α-peptides can form helices in which the H-bond directionality alternates along the backbone because neighboring amide groups are oriented in opposite directions. Alternating H-bond directions are seen also in helices formed by unnatural peptidic backbones, e.g., those containing ß- or γ-amino acid residues. In the present study, we used NMR spectroscopy and crystallography to evaluate the conformational preferences of the novel γ-amino acid (1R,2R,3S)-2-(1-aminopropyl)-cyclohexanecarboxylic acid (APCH), which is constrained by a six-membered ring across its Cα-Cß bond. These studies were made possible by the development of a stereoselective synthesis of N-protected APCH. APCH strongly enforces the α/γ-peptide 12/10-helical secondary structure, which features alternating H-bond directionality. Thus, APCH residues appear to have a conformational propensity distinct from those of other cyclically constrained γ-amino acid residues.

Evaluation of a cyclopentane-based γ-amino acid for the ability to promote α/γ-peptide secondary structure.

We report the asymmetric synthesis of the γ-amino acid (1R,2R)-2-aminomethyl-1-cyclopentane carboxylic acid (AMCP) and an evaluation of this residue's potential to promote secondary structure in α/γ-peptides. Simulated annealing calculations using NMR-derived distance restraints obtained for α/γ-peptides in chloroform reveal that AMCP-containing oligomers are conformationally flexible. However, additional evidence suggests that an internally hydrogen-bonded helical conformation is partially populated in solution. From these data, we propose characteristic NOE patterns for the formation of the α/γ-peptide 12/10-helix and discuss the apparent conformational frustration of AMCP-containing oligomers.

Parallel ß-sheet vibrational couplings revealed by 2D IR spectroscopy of an isotopically labeled macrocycle: quantitative benchmark for the interpretation of amyloid and protein infrared spectra.

Infrared spectroscopy is playing an important role in the elucidation of amyloid fiber formation, but the coupling models that link spectra to structure are not well tested for parallel ß-sheets. Using a synthetic macrocycle that enforces a two stranded parallel ß-sheet conformation, we measured the lifetimes and frequency for six combinations of doubly (13)C═(18)O labeled amide I modes using 2D IR spectroscopy. The average vibrational lifetime of the isotope labeled residues was 550 fs. The frequencies of the labels ranged from 1585 to 1595 cm(-1), with the largest frequency shift occurring for in-register amino acids. The 2D IR spectra of the coupled isotope labels were calculated from molecular dynamics simulations of a series of macrocycle structures generated from replica exchange dynamics to fully sample the conformational distribution. The models used to simulate the spectra include through-space coupling, through-bond coupling, and local frequency shifts caused by environment electrostatics and hydrogen bonding. The calculated spectra predict the line widths and frequencies nearly quantitatively. Historically, the characteristic features of ß-sheet infrared spectra have been attributed to through-space couplings such as transition dipole coupling. We find that frequency shifts of the local carbonyl groups due to nearest neighbor couplings and environmental factors are more important, while the through-space couplings dictate the spectral intensities. As a result, the characteristic absorption spectra empirically used for decades to assign parallel ß-sheet secondary structure arises because of a redistribution of oscillator strength, but the through-space couplings do not themselves dramatically alter the frequency distribution of eigenstates much more than already exists in random coil structures. Moreover, solvent exposed residues have amide I bands with >20 cm(-1) line width. Narrower line widths indicate that the amide I backbone is solvent protected inside the macrocycle. This work provides calculated and experimentally verified couplings for parallel ß-sheets that can be used in structure-based models to simulate and interpret the infrared spectra of ß-sheet containing proteins and protein assemblies, such as amyloid fibers.

Parallel ß-sheet secondary structure is stabilized and terminated by interstrand disulfide cross-linking.

Disulfide bonds between Cys residues in adjacent strands of parallel ß-sheets are rare among proteins, which suggests that parallel ß-sheet structure is not stabilized by such disulfide cross-links. We report experimental results that show, surprisingly, that an interstrand disulfide bond can stabilize parallel ß-sheets formed by an autonomously folding peptide in aqueous solution. NMR analysis reveals that parallel ß-sheet structure is terminated beyond the disulfide bond, which causes deviation from the extended backbone conformation at one of the Cys residues.

Helix formation in preorganized beta/gamma-peptide foldamers: hydrogen-bond analogy to the alpha-helix without alpha-amino acid residues.

We report the first high-resolution structural data for the beta/gamma-peptide 13-helix (i,i+3 C=O...H-N H-bonds), a secondary structure that is formed by oligomers with a 1:1 alternation of beta- and gamma-amino acid residues. Our characterization includes both crystallographic and 2D NMR data. Previous studies suggested that beta/gamma-peptides constructed from conformationally flexible residues adopt a different helical secondary structure in solution. Our design features preorganized beta- and gamma-residues, which strongly promote 13-helical folding by the 1:1 beta/gamma backbone.

Stereospecific synthesis of conformationally constrained gamma-amino acids: new foldamer building blocks that support helical secondary structure.

A highly stereoselective synthesis of novel cyclically constrained gamma-amino acid residues is presented. The key step involves organocatalytic Michael addition of an aldehyde to 1-nitrocyclohexene. After aldehyde reduction, this approach provides optically active beta-substituted delta-nitro alcohols (96-99% ee), which can be converted to gamma-amino acid residues with a variety of substituents at the alpha position. We have used these new building blocks to prepare alpha/gamma-peptide foldamers that adopt a specific helical conformation in solution and in the solid state.