Impact of γ‑Amino Acid Residue Preorganization on α/γ-Peptide Foldamer Helicity in Aqueous Solution

α/γ-Peptide foldamers containing either γ⁴-amino acid residues or ring-constrained γ-amino acid residues have been reported to adopt 12-helical secondary structure in nonpolar solvents and in the solid state. These observations have engendered speculation that the seemingly flexible γ⁴ residues have a high intrinsic helical propensity and that residue-based preorganization may not significantly stabilize the 12-helical conformation. However, the prior studies were conducted in environments that favor intramolecular H-bond formation. Here, we use 2D-NMR to compare the ability of γ⁴ residues and cyclic γ residues to support 12-helix formation in more challenging environments, methanol and water. Both γ residue types support 12-helical folding in methanol, but only the cyclically constrained γ residues promote helicity in water. These results demonstrate the importance of residue-based preorganization strategies for achieving stable folding among short foldamers in aqueous solution

Impact of Backbone Pattern and Residue Substitution on Helicity in α/β/γ-Peptides

We have evaluated the impact of changes in the chemical structure of peptidic oligomers containing α-, β-, and γ-amino acid residues (α/β/γ-peptides) on the propensities of these oligomers to adopt helical conformations in aqueous and alcoholic solutions. These studies were inspired by our previous discovery that α/β/γ-peptides containing a regular αγααβα hexad repeat adopt an α-helix-like conformation in which the β and γ residues are aligned in a stripe along one side, and the remainder of the helix surface is defined by the α residues. This helix was found to be most stable when the β and γ residues were rigidified with specific cyclic constraints. Relaxation of the β residue constraints caused profound conformational destabilization, but relaxation of the γ residue constraints led to only a moderate drop in helicity. The new work more broadly characterizes the effect of γ residue substitution on helix stability, based on circular dichroism and two-dimensional NMR measurements. We find that even a fully unsubstituted γ residue (derived from γ-aminobutyric acid) supports a moderate helical propensity, which is surprising in light of the strong destabilizing effect of glycine residues on α-helix stability. Additional studies examine the effects of altering sequence in terms of amino acid type, by comparing a prototype with the αγααβα hexad pattern to isomers with irregular arrangements of the α, β, and γ residues along the backbone. The data indicate that the strong helix-forming propensity previously discovered for α/β/γ-peptide 12-mers is retained when sequence is varied, with small variations detected across diverse α-β-γ placements. These structural findings suggest that α/β/γ-peptide scaffolds represent versatile scaffolds for the design of peptidic foldamers that display specific functions

Parallel β-Sheet Secondary Structure Is Stabilized and Terminated by Interstrand Disulfide Cross-Linking

Disulfide bonds between Cys residues in adjacent strands of parallel β-sheets are rare among proteins, which suggests that parallel β-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 β-sheets formed by an autonomously folding peptide in aqueous solution. NMR analysis reveals that parallel β-sheet structure is terminated beyond the disulfide bond, which causes deviation from the extended backbone conformation at one of the Cys residues

Polymer Chain Length Effects on Fibroblast Attachment on Nylon-3-Modified Surfaces

Nylon-3 polymers have a polyamide backbone reminiscent of that found in proteins (β- vs α-amino acid residues, respectively), which makes these materials interesting for biological applications. Because of the versatility of the ring-opening polymerization process and the variety of β-lactam starting materials available, the structure of nylon-3 copolymers is highly amenable to alteration. A previous study showed that relatively subtle changes in the structure or ratio of hydrophobic and cationic subunits that comprise these polymers can result in significant changes in the ability of nylon-3-bearing surfaces to support cell adhesion and spreading. In the present study, we have exploited the highly tailorable nature of these polymers to synthesize new versions possessing a wide range of chain lengths, with the intent of optimizing these materials for use as cell-supportive substrates. We find that longer nylon-3 chains lead to better fibroblast attachment on modified surfaces and that at the optimal chain lengths less hydrophobic subunits are superior. The best polymers we identified are comparable to an RGD-containing peptide in supporting fibroblast attachment. The results described here will help to focus future efforts aimed at refining nylon-3 copolymer substrates for specific tissue engineering applications

Inhibition of Ice Recrystallization by Nylon‑3 Polymers

Nontoxic cryoprotectants are needed for storage of tissues and food preservation. Frozen tissue is particularly susceptible to damage caused by formation of large ice crystals during the thawing process. The current practice of using 5 wt % DMSO for cryopreservation does not produce 100% cell viability post-thaw, at least in part because of DMSO toxicity that is manifested during the freezing and thawing stages of the process. Recently, poly­(vinyl alcohol) (PVA) has shown promise in inhibiting ice recrystallization, an activity that is critical for cryoprotection. Inspired by this discovery, we have evaluated nylon-3 polymers for ice recrystallization inhibition activity and for toxicity toward mammalian cells. A survey of homo- and heteropolymers, with side chains bearing variable functionality, has identified new nylon-3 materials that display excellent ice recrystallization inhibition activity and low toxicity

Helix Propensities of Amino Acid Residues via Thioester Exchange

We describe the use of thioester exchange equilibria to measure the propensities of amino acid residues to participate in helical secondary structure at room temperature in the absence of denaturants. Thermally or chemically induced unfolding has previously been employed to measure α-helix propensities among proteinogenic α-amino acid residues, and quantitative comparison with precedents indicates that the thioester exchange system is reliable for residues that lack side chain charge. This system allows the measurement of α-helix propensities for d-α-amino acid residues and propensities of residues with nonproteinogenic backbones, such as those derived from a β-amino acid, to participate in an α-helix-like secondary structure

Nylon‑3 Polymers That Enable Selective Culture of Endothelial Cells

Substrates that selectively encourage the growth of specific cell types are valuable for the engineering of complex tissues. Some cell-selective peptides have been identified from extracellular matrix proteins; these peptides have proven useful for biomaterials-based approaches to tissue repair or regeneration. However, there are very few examples of synthetic materials that display selectivity in supporting cell growth. We describe nylon-3 polymers that support in vitro culture of endothelial cells but do not support the culture of smooth muscle cells or fibroblasts. These materials may be promising for vascular biomaterials applications

Evaluation of the Ser-His Dipeptide, a Putative Catalyst of Amide and Ester Hydrolysis

Efficient hydrolysis of amide bonds has long been a reaction of interest for organic chemists. The rate constants of proteases are unmatched by those of any synthetic catalyst. It has been proposed that a dipeptide containing serine and histidine is an effective catalyst of amide hydrolysis, based on an apparent ability to degrade a protein. The capacity of the Ser-His dipeptide to catalyze the hydrolysis of several discrete ester and amide substrates is investigated using previously described conditions. This dipeptide does not catalyze the hydrolysis of amide or unactivated ester groups in any of the substrates under the conditions evaluated

Single-Cell, Time-Resolved Antimicrobial Effects of a Highly Cationic, Random Nylon‑3 Copolymer on Live <i>Escherichia coli</i>

Synthetic random copolymers based on the nylon-3 (β-peptide) backbone show promise as inexpensive antimicrobial agents resistant to proteolysis. We present a time-resolved observational study of the attack of a particular copolymer <b>MM</b>_<b>63</b><b>:CHx</b>_<b>37</b> on single, live <i>Escherichia coli</i> cells. The composition and chain length of <b>MM</b>_<b>63</b><b>:CHx</b>_<b>37</b> (63% cationic subunits, 37% hydrophobic subunits, 35-subunit average length) were optimized to enhance antibacterial activity while minimizing lysis of human red blood cells. For <i>E. coli</i> cells that export GFP to the periplasm, we obtain alternating phase-contrast and green fluorescence images with a time resolution of 12 s over 60 min following initiation of copolymer flow. Within seconds, cells shrink and exhibit the same plasmolysis spaces that occur following abrupt external osmotic upshift. The osmoprotection machinery attempts to replenish cytoplasmic water, but recovery is interrupted by permeabilization of the cytoplasmic membrane (CM) to GFP. Evidently, the highly cationic copolymer and its counterions rapidly translocate across the outer membrane without permeabilizing it to GFP. The CM permeabilization event is spatially localized. Cells whose CM has been permeabilized never recover growth. The minimum inhibitory concentration (MIC) for cells lacking the osmolyte importer ProP is 4-fold smaller than for normal cells, suggesting that osmoprotection is an important survival strategy. In addition, at the time of CM permeabilization, we observe evidence of oxidative stress. The MIC under anaerobic conditions is at least 8-fold larger than under aerobic conditions, further implicating oxidative damage as an important bacteriostatic effect. Once the copolymer reaches the periplasm, multiple growth-halting mechanisms proceed in parallel

Medium Effects on Minimum Inhibitory Concentrations of Nylon-3 Polymers against <i>E. coli</i>

<div><p>Minimum inhibitory concentrations (MICs) against <i>E. coli</i> were measured for three nylon-3 polymers using Luria-Bertani broth (LB), brain-heart infusion broth (BHI), and a chemically defined complete medium (EZRDM). The polymers differ in the ratio of hydrophobic to cationic subunits. The cationic homopolymer is inert against <i>E. coli</i> in BHI and LB, but becomes highly potent in EZRDM. A mixed hydrophobic/cationic polymer with a hydrophobic <i>t</i>-butylbenzoyl group at its N-terminus is effective in BHI, but becomes more effective in EZRDM. Supplementation of EZRDM with the tryptic digest of casein (often found in LB) recapitulates the LB and BHI behavior. Additional evidence suggests that polyanionic peptides present in LB and BHI may form electrostatic complexes with cationic polymers, decreasing activity by diminishing binding to the anionic lipopolysaccharide layer of <i>E. coli</i>. In contrast, two natural antimicrobial peptides show no medium effects. Thus, the use of a chemically defined medium helps to reveal factors that influence antimicrobial potency of cationic polymers and functional differences between these polymers and evolved antimicrobial peptides.</p></div