Longer charged amino acids favor ß-strand formation in hairpin peptides.

Interactions between charged amino acids significantly influence the structure and function of proteins. The encoded charged amino acids Asp, Glu, Arg, and Lys have different number of hydrophobic methylenes linking the backbone to the charged functionality. It remains to be fully understood how does this difference in the number of methylenes affect protein structure stability. Protein secondary structures are the fundamental three-dimensional building blocks of protein structures. ß-Sheet structures are particularly interesting, because these structures have been associated with a number of protein misfolding diseases. Herein, we report the effect of charged amino acid side chain length at two ß-strand positions individually on the stability of a ß-hairpin. The charged amino acids include side chains with a carboxylate, an ammonium, or a guanidinium group. The experimental peptides, fully folded reference peptides, and fully unfolded reference peptides were synthesized by solid phase peptide synthesis and analyzed by 2D NMR methods including TOCSY, DQF-COSY, and ROESY. Sequence specific assignments were performed for all peptides. The chemical shift data were used to derive the fraction folded population and the folding free energy for the experimental peptides. Results showed that the fraction folded population increased with increasing charged amino acid side chain length. These results should be useful for developing functional peptides that adopt the ß-conformation.

Swapping the Positions in a Cross-Strand Lateral Ion-Pairing Interaction between Ammonium- and Carboxylate-Containing Residues in a ß-Hairpin.

Cross-strand lateral ion-pairing interactions are important for antiparallel ß-sheet stability. Statistical studies suggested that swapping the position of cross-strand lateral residues should not significantly affect the interaction. Herein, we swapped the position of ammonium- and carboxylate-containing residues with different side-chain lengths in a cross-strand lateral ion-pairing interaction in a ß-hairpin. The peptides were analyzed by 2D-NMR. The fraction folded population and folding free energy were derived from the chemical shift data. The ion-pairing interaction energy was derived using double mutant cycle analysis. The general trends for the fraction folded population and interaction energetics remained similar upon swapping the position of the interacting charged residues. The most stabilizing cross-strand interactions were between short residues, similar to the unswapped study. However, the fraction folded populations for most of the swapped peptides were higher compared to the corresponding unswapped peptides. Furthermore, subtle differences in the ion-pairing interaction energy upon swapping were observed, most likely due to the "unleveled" relative positioning of the interacting residues created by the inherent right-handed twist of the structure. These results should be useful for developing functional peptides that rely on lateral ion-pairing interactions across antiparallel ß-strands.

Using 19F NMR to Investigate Cationic Carbon Dot Association with Per- and Polyfluoroalkyl Substances (PFAS).

There is much concern about per- and polyfluoroalkyl substances (PFAS) based on their environmental persistence and toxicity, resulting in an urgent need for remediation technologies. This study focused on determining if nanoscale polymeric carbon dots are a viable sorbent material for PFAS and developing fluorine nuclear magnetic resonance spectroscopy (19F NMR) methods to probe interactions between carbon dots and PFAS at the molecular scale. Positively charged carbon dots (PEI-CDs) were synthesized using branched polyethyleneimine to target anionic PFAS by promoting electrostatic interactions. PEI-CDs were exposed to perfluorooctanoic acid (PFOA) to assess their potential as a PFAS sorbent material. After exposure to PFOA, the average size of the PEI-CDs increased (1.6 ± 0.5 to 7.8 ± 1.8 nm) and the surface charge decreased (+38.6 ± 1.1 to +26.4 ± 0.8 mV), both of which are consistent with contaminant sorption. 19F NMR methods were developed to gain further insight into PEI-CD affinity toward PFAS without any complex sample preparation. Changes in PFOA peak intensity and chemical shift were monitored at various PEI-CD concentrations to establish binding curves and determine the chemical exchange regime. 19F NMR spectral analysis indicates slow-intermediate chemical exchange between PFOA and CDs, demonstrating a high-affinity interaction. The α-fluorine had the greatest change in chemical shift and highest affinity, suggesting electrostatic interactions are the dominant sorption mechanism. PEI-CDs demonstrated affinity for a wide range of analytes when exposed to a mixture of 24-PFAS, with a slight preference toward perfluoroalkyl sulfonates. Overall, this study shows that PEI-CDs are an effective PFAS sorbent material and establishes 19F NMR as a suitable method to screen for novel sorbent materials and elucidate interaction mechanisms.

Structural impact of thioamide incorporation into a ß-hairpin.

The thioamide is a naturally-occurring single atom substitution of the canonical amide bond. The exchange of oxygen to sulfur alters the amide's physical and chemical characteristics, thereby expanding its functionality. Incorporation of thioamides in prevalent secondary structures has demonstrated that they can either have stabilizing, destabilizing, or neutral effects. We performed a systematic investigation of the structural impact of thioamide incorporation in a ß-hairpin scaffold with nuclear magnetic resonance (NMR). Thioamides as hydrogen bond donors did not increase the foldedness of the more stable "YKL" variant of this scaffold. In the less stable "HPT" variant of the scaffold, the thioamide could be stabilizing as a hydrogen bond donor and destabilizing as a hydrogen bond acceptor, but the extent of the perturbation depended upon the position of incorporation. To better understand these effects we performed structural modelling of the macrocyclic folded HPT variants. Finally, we compare the thioamide effects that we observe to previous studies of both side-chain and backbone perturbations to this ß-hairpin scaffold to provide context for our observations.