The formation of ordered cross-β amyloid protein aggregates is associated with a variety of human disorders. While conventional infrared methods serve as sensitive reporters of the presence of these amyloids, the recently discovered amyloid secondary structure of cross-α fibrils presents new questions and challenges. Herein, we report results using Fourier transform infrared spectroscopy and two-dimensional infrared spectroscopy to monitor the aggregation of one such cross-α–forming peptide, phenol soluble modulin alpha 3 (PSMα3). Phenol soluble modulins (PSMs) are involved in the formation and stabilization of Staphylococcus aureus biofilms, making sensitive methods of detecting and characterizing these fibrils a pressing need. Our experimental data coupled with spectroscopic simulations reveals the simultaneous presence of cross-α and cross-β polymorphs within samples of PSMα3 fibrils. We also report a new spectroscopic feature indicative of cross-α fibrils.

Cation effects on proteins have been a challenge to understand. Herein, we present two-dimensional infrared (2DIR) spectroscopic measurements, coupled with molecular dynamics and spectroscopic calculations, of N-methylacetamide (NMA), a common model of the peptide backbone, in aqueous CaCl 2 . The 2DIR spectra reveal that the dynamics of the amide carbonyl of NMA is dominated by exchange between two states of varying hydration, one possessing a structure similar to aqueous NMA and one that is dehydrated by one hydrogen bond. In addition, we demonstrate that at high (>5 M) CaCl 2 concentrations, direct binding of Ca 2+ to the carbonyl of NMA occurs, stabilizing an iminium-type resonance structure of NMA, with a characteristic CN + stretch frequency at 1680 cm −1 . This species is only marginally populated and is only detectable in 2DIR spectra due to its larger transition strength.

Vibrational Stark shifts were explored in aqueous solutions of organic molecules with carbonyl-and nitrile-containing constituents. In many cases, the vibrational resonances from these moieties shifted toward lower frequency as salt was introduced into solution. This is in contrast to the blue-shift that would be expected based upon Onsager's reaction field theory. Salts containing well-hydrated cations like Mg 2+ or Li + led to the most pronounced Stark shift for the carbonyl group, while poorly hydrated cations like Cs + had the greatest impact on nitriles. Moreover, salts containing I − gave rise to larger Stark shifts than those containing Cl − . Molecular dynamics simulations indicated that cations and anions both accumulate around the probe in an ion-and probe-dependent manner. An electric field was generated by the ion pair, which pointed from the cation to the anion through the vibrational chromophore. This resulted from solvent-shared binding of the ions to the probes, consistent with their positions in the Hofmeister series. The "anti-Onsager" Stark shifts occur in both vibrational spectroscopy and fluorescence measurements.

The formation of ordered cross-β amyloid protein aggregates is associated with a variety of human disorders. While conventional infrared methods serve as sensitive reporters of the presence of these amyloids, the recently discovered amyloid secondary structure of cross- fibrils presents new questions and challenges. Herein, we report results using Fourier Transform infrared (FTIR) spectroscopy and two-dimensional infrared (2DIR) spectroscopy, to monitor the aggregation of one such cross-alpha forming peptide, phenol soluble modulin alpha 3 (PSM⍺3). Phenol soluble modulins (PSMs) are involved in the formation and stabilization of Staphylococcus aureus biofilms, making sensitive methods of detecting and characterizing these fibrils a pressing need. Our experimental data, coupled with spectroscopic simulations, reveals the simultaneous presence of cross-⍺ and cross-β polymorphs within samples of PSM⍺3 fibrils. We also report a new spectroscopic feature indicative of cross-alpha fibrils.

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