2020, Scheerer, David, Chi, Heng, McElheny, Dan, Keiderling, Timothy A., Hauser, Karin

Site-specific isotopic labeling of molecules is a widely used approach in IR-spectroscopy to resolve local contributions to vibrational modes. The induced frequency shift of the corresponding IR band depends on the substituted masses, but also on hydrogen bonding and on vibrational coupling. The impact of these different factors was analyzed with a designed three-stranded β-sheet peptide and by use of selected 13C isotope substitutions at multiple positions in the peptide backbone. Single strand labels give rise to isotopically shifted bands at different frequencies depending on the specific sites, demonstrating sensitivity to the local environment. Cross-strand double and triple labeled peptides exhibited two resolved bands, which could be uniquely assigned to specific residues, whose equilibrium IR indicated only weak local-mode coupling. Temperature-jump IR-laser spectroscopy was applied to monitor structural dynamics and revealed an impressive enhancement of the isotope sensitivity to both local positions and coupling between them as compared to equilibrium FTIR. Site-specific relaxation rates were altered upon introduction of additional cross-strand isotopes. Likewise, the rates for the global β-sheet dynamics were affected in a manner dependent on the distinct relaxation behavior of the labeled oscillator. The study demonstrates that isotope labels do not just provide local structural probes, but they rather sense the dynamic complexity of the molecular environment.

2018-01-18, Scheerer, David, Chi, Heng, McElheny, Dan, Samer, Ayesha, Keiderling, Timothy A., Hauser, Karin

A series of closely related peptide sequences that form triple-strand structures was designed with a variation of cross-strand aromatic interactions and spectroscopically studied as models for β-sheet formation and stabilities. Structures of the three-strand models were determined with NMR methods and temperature-dependent equilibrium studies performed using circular dichroism and Fourier transform infrared spectroscopies. Our equilibrium data show that the presence of a direct cross-strand aromatic contact in an otherwise folded peptide does not automatically result in an increased thermal stability and can even distort the structure. The effect on the conformational dynamics was studied with infrared-detected temperature-jump relaxation methods and revealed a high sensitivity to the presence and the location of the aromatic cross-links. Aromatic contacts in the three-stranded peptides slow down the dynamics in a site-specific manner, and the impact seems to be related to the distance from the turn. With a Xxx-^DPro linkage as a probe with some sensitivity for the turn, small differences were revealed in the relative relaxation of the sheet strands and turn regions. In addition, we analyzed the component hairpins, which showed less uniform dynamics as compared to the parent three-stranded β-sheet peptides.

2018-11-21, Scheerer, David, Chi, Heng, McElheny, Dan, Keiderling, Timothy A., Hauser, Karin

2017-02, Keiderling, Timothy A., Chi, Heng, McElheny, Dan, Walker, Allen, Scheerer, David, Hauser, Karin

Spectroscopic studies of peptide β-sheets is complicated by their having many forms (parallel vs. anti-parallel, twisted or flat, in and out of register) and being subject to aggregation so that the molecular state being studied experimentally becomes obscure and thus very difficult to model theoretically. We have used simple hairpin models (strand-turn-strand) to develop monomer structures that have cross-strand anti-parallel H-bonding characteristic of sheets, however these strands are also solvated on each edge.

2018-02, Keiderling, Timothy A., Chi, Heng, McElheny, Dan, Scheerer, David, Samer, Ayesha, Hauser, Karin, Vazquez, Frank

b-sheets can be modeled as antiparallel strands coupled by turns. Isolated model systems are less stable, due to amide solvation), but can be induced to fold into sheet-like structures by restricting the conformational space of turn residues, such as with DPro-Gly sequences, or by inclusion of crossstrand hydrophobic interactions, especially aromatic contacts, which tend to desolvate parts of the structure. Expanding to multistrand structures can provide different characteristics for study of physical properties. Larger structures have strand differentiation, since the center strand of a threestrand sheet has a different H-bonds than the outer ones. In larger structures, integral hairpin-like sequences of either the first two or the second two strands might have different stabilities and folding mechanisms, if it is overall a multistate process. To explore these factors, we have prepared a series of three-stranded sheet structures that are stabilized by either DPro-Gly or by Aib-Gly turns and contain various Trp-Tyr side-chain contacts. To assess the impact of the third strand we also separately synthesized the component hairpins and studied their relative properties with temperature dependent circular dichroism, fluorescence, infrared (IR) and vibrational circular dichroism for evaluation of global thermodynamic equilibria as well as IR–detected temperature jump relaxation dynamics of selected components (strands, turns). Where possible, NMR structures were obtained for comparison and MD simulations of unfolding were used to aid interpretations. While all the sequences showed evidence of b-structure formation, the extent and stability varied markedly. In terms of equilibrium properties, the turn residues (especially DPro-Gly) seemed to have the strongest influence, while the aromatic contacts had minor effects. However in terms of dynamics, the aromatic effects differentiated the structures more, suggesting a variation in the folding mechanisms.

2016, Popp, Alexander, Scheerer, David, Chi, Heng, Keiderling, Timothy A., Hauser, Karin

Turn residues as well as side-chain interactions play an important role for the folding of ß-sheets. We investigated the conformational dynamics of a three-stranded ß-sheet peptide (^DP^DP) and a two-stranded ß-hairpin (WVYY-^DP) by time-resolved temperature-jump infrared spectroscopy. Both peptide sequences contain ^DPro-Gly residues that favor a tight ß-turn. The three-stranded ß-sheet (Ac-VFITS^DPGKTYTEV^DPGOKILQ-NH₂) is stabilized by the turn sequences, whereas the ß-hairpin (SWTVE^DPGKYTYK-NH₂) folding is assisted both by the turn sequence and by hydrophobic cross-strand interactions. Relaxation times after the T-jump were monitored as a function of temperature and occur on a sub-microsecond time scale, ^DP^DP being faster than WVYY-^DP. The Xxx-^DPro tertiary amide provides a detectable IR band allowing us to site-specifically probe the dynamics. The relative importance of the turn versus the intra-strand stability in ß-sheet formation is discussed.