Simultaneously Measured Kinetics of Two Amyloid Polymorphs Using Cross Peak Specific 2D IR Spectroscopy.

The origin of in vitro amyloid fibril polymorphs is debated, in part, because few techniques can simultaneously monitor the formation kinetics of multiple amyloid polymorphs. Using a cross-peak specific polarization scheme, ⟨0°,0°,60°,-60°⟩, we resolve 22 previously unseen cross peaks in the 2D IR spectra of amyloid fibrils formed by the human islet amyloid polypeptide (hIAPP). Those cross peaks include a subset assigned to a second fibril polymorph, which forms on a slower time scale. We simulated the data with three different kinetic models for polymorph formation. Only a model based on secondary nucleation reproduces the cross peak kinetics. These experiments are evidence that fibrils formed by secondary nucleation have a different polymorphic structure than the parent fibrils and illustrate the enhanced structural resolution of this new cross peak specific polarization scheme.

Phase stable, shot-to-shot measurement of third- and fifth-order two-quantum correlation spectra using a pulse shaper in the pump-probe geometry.

We demonstrate the first phase stable measurement of a third-order 2Q spectrum using a pulse shaper in the pump-probe geometry. This measurement was achieved by permuting the time-ordering of the pump pulses, thus rearranging the signal pathways that are emitted in the probe direction. The third-order 2Q spectrum is self-heterodyned by the probe pulse. Using this method, one can interconvert between a 1Q experiment and a 2Q experiment by simply reprogramming a pulse shaper or delay stage. We also measure a fifth-order absorptive 2Q spectrum in the pump-probe geometry, which contains similar information as a third-order experiment but does not suffer from dispersive line shapes. To do so, we introduce methods to minimize saturation-induced artifacts of the pulse shaper, improving fifth-order signals. These techniques add new capabilities for 2D spectrometers that use pulse shapers in the pump-probe beam geometry.

A polarization scheme that resolves cross-peaks with transient absorption and eliminates diagonal peaks in 2D spectroscopy.

Two-dimensional (2D) optical spectroscopy contains cross-peaks that are helpful features for determining molecular structure and monitoring energy transfer, but they can be difficult to resolve from the much more intense diagonal peaks. Transient absorption (TA) spectra contain transitions similar to cross-peaks in 2D spectroscopy, but in most cases they are obscured by the bleach and stimulated emission peaks. We report a polarization scheme, <0°,0°,+θ2(t2),-θ2(t2)>, that can be easily implemented in the pump-probe beam geometry, used most frequently in 2D and TA spectroscopy. This scheme removes the diagonal peaks in 2D spectroscopies and the intense bleach/stimulated emission peaks in TA spectroscopies, thereby resolving the cross-peak features. At zero pump-probe delay, θ2 = 60° destructively interferes two Feynman paths, eliminating all signals generated by field interactions with four parallel transition dipoles, and the intense diagonal and bleach/stimulated emission peaks. At later delay times, θ2(t2) is adjusted to compensate for anisotropy caused by rotational diffusion. When implemented with TA spectroscopy or microscopy, the pump-probe spectrum is dominated by the cross-peak features. The local oscillator is also attenuated, which enhances the signal two times. This overlooked polarization scheme reduces spectral congestion by eliminating diagonal peaks in 2D spectra and enables TA spectroscopy to measure similar information given by cross-peaks in 2D spectroscopy.

Analysis of amyloid-like secondary structure in the Cryab-R120G knock-in mouse model of hereditary cataracts by two-dimensional infrared spectroscopy.

αB-crystallin is a small heat shock protein that forms a heterooligomeric complex with αA-crystallin in the ocular lens. It is also widely distributed in tissues throughout the body and has been linked with neurodegenerative diseases such as Alzheimer's, where it is associated with amyloid fibrils. Crystallins can form amorphous aggregates in cataracts as well as more structured amyloid-like fibrils. The arginine 120 to glycine (R120G) mutation in αB-crystallin (Cryab-R120G) results in high molecular weight crystallin protein aggregates and loss of the chaperone activity of the protein in vitro, and it is associated with human hereditary cataracts and myopathy. Characterizing the amorphous (unstructured) versus the highly ordered (amyloid fibril) nature of crystallin aggregates is important in understanding their role in disease and important to developing pharmacological treatments for cataracts. We investigated protein secondary structure in wild-type (WT) and Cryab-R120G knock-in mutant mouse lenses using two-dimensional infrared (2DIR) spectroscopy, which has been used to detect amyloid-like fibrils in human lenses and measure UV radiation-induced changes in porcine lenses. Our goal was to compare the aggregated proteins in this mouse lens model to human lenses and evaluate the protein structural relevance of the Cryab-R120G knock-in mouse model to general age-related cataract disease. In the 2DIR spectra, amide I diagonal peak frequencies were red-shifted to smaller wavenumbers in mutant mouse lenses as compared to WT mouse lenses, consistent with an increase in ordered secondary structure. The cross peak frequency and intensity indicated the presence of amyloid in the mutant mouse lenses. While the diagonal and cross peak changes in location and intensity from the 2DIR spectra indicated significant structural differences between the wild type and mutant mouse lenses, these differences were smaller than those found in human lenses; thus, the Cryab-R120G knock-in mouse lenses contain less amyloid-like secondary structure than human lenses. The results of the 2DIR spectroscopy study confirm the presence of amyloid-like secondary structure in Cryab-R120G knock-in mice with cataracts and support the use of this model to study age-related cataract.

Shot-to-shot 2D IR spectroscopy at 100 kHz using a Yb laser and custom-designed electronics.

The majority of 2D IR spectrometers operate at 1-10 kHz using Ti:Sapphire laser technology. We report a 2D IR spectrometer designed around Yb:KGW laser technology that operates shot-to-shot at 100 kHz. It includes a home-built OPA, a mid-IR pulse shaper, and custom-designed electronics with optional on-chip processing. We report a direct comparison between Yb:KGW and Ti:Sapphire based 2D IR spectrometers. Even though the mid-IR pulse energy is much lower for the Yb:KGW driven system, there is an 8x improvement in signal-to-noise over the 1 kHz Ti:Sapphire driven spectrometer to which it is compared. Experimental data is shown for sub-millimolar concentrations of amides. Advantages and disadvantages of the design are discussed, including thermal background that arises at high repetition rates. This fundamental spectrometer design takes advantage of newly available Yb laser technology in a new way, providing a straightforward means of enhancing sensitivity.

Heavy-Atom-Substituted Nucleobases in Photodynamic Applications: Substitution of Sulfur with Selenium in 6-Thioguanine Induces a Remarkable Increase in the Rate of Triplet Decay in 6-Selenoguanine.

Sulfur substitution of carbonyl oxygen atoms of DNA/RNA nucleobases promotes ultrafast intersystem crossing and near-unity triplet yields that are being used for photodynamic therapy and structural-biology applications. Replacement of sulfur with selenium or tellurium should significantly red-shift the absorption spectra of the nucleobases without sacrificing the high triplet yields. Consequently, selenium/tellurium-substituted nucleobases are thought to facilitate treatment of deeper tissue carcinomas relative to the sulfur-substituted analogues, but their photodynamics are yet unexplored. In this contribution, the photochemical relaxation mechanism of 6-selenoguanine is elucidated and compared to that of the 6-thioguanine prodrug. Selenium substitution leads to a remarkable enhancement of the intersystem crossing lifetime both to and from the triplet manifold, resulting in an efficiently populated, yet short-lived triplet state. Surprisingly, the rate of triplet decay in 6-selenoguanine increases by 835-fold compared to that in 6-thioguanine. This appears to be an extreme manifestation of the classical heavy-atom effect in organic photochemistry, which challenges conventional wisdom.