Quantitative Spectral Analysis of Coherent Anti-Stokes Raman Scattering Signals: C–H Stretching Modes of the Methyl Group

Coherent anti-Stokes Raman scattering (CARS) vibrational spectroscopy has been extensively developed into a powerful analytical technique to study various molecules. Quantitative interpretation of CARS spectra can help to improve CARS for chemical analysis and extend its analytical applications. In this work, we quantitatively analyzed CARS signals originating from the methyl groups in poly­(dimethylsiloxane) (PDMS), with the help of the bond additivity method. Experimentally, a home-built CARS spectrometer modified from a commercial sum frequency generation spectrometer was used to collect CARS spectra from a PDMS film. Theoretically, we successfully reproduced the peak intensity ratio of C–H symmetric and asymmetric stretching modes of the PDMS methyl group in different polarization combinations based on bond additivity method and Raman depolarization ratio. This research shows that bond additivity theory can help to obtain the third-order nonlinear susceptibility tensor properties probed by different polarization combinations used in CARS spectroscopy. The method developed in this work could also be applied to CARS vibrational stretching analysis of other functional groups, providing quantitative understanding of CARS spectrum for applications in spectroscopy

Elementary Reactions in the Functional Triads of the Blue-Light Photoreceptor BLUF Domain

The blue light using the flavin (BLUF) domain is one of the smallest photoreceptors in nature, which consists of a unique bidirectional electron-coupled proton relay process in its photoactivation reaction cycle. This perspective summarizes our recent efforts in dissecting the photocycle into three elementary processes, including proton-coupled electron transfer (PCET), proton rocking, and proton relay. Using ultrafast spectroscopy, we have determined the temporal sequence, rates, kinetic isotope effects (KIEs), and concertedness of these elementary steps. Our findings provide important implications for illuminating the photoactivation mechanism of the BLUF domain and suggest an engineering platform to characterize intricate reactions involving proton motions that are ubiquitous in nonphotosensitive protein machines

Possible Existence of α‑Sheets in the Amyloid Fibrils Formed by a TTR_105–115 Mutant

Herein, we combine several methods to characterize the fibrils formed by a TTR_105–115 mutant in which Leu111 is replaced by the unnatural amino acid aspartic acid 4-methyl ester. We find that this mutant peptide exhibits significantly different aggregation behavior than the wild-type peptide: (1) it forms fibrils with a much faster rate, (2) its fibrils lack the long-range helical twists observed in TTR_105–115 fibrils, (3) its fibrils exhibit a giant far-UV circular dichroism signal, and (4) its fibrils give rise to an unusual amide I′ band consisting of four distinct and sharp peaks. On the basis of these results and also several previous computational studies, we hypothesize that the fibrils formed by this TTR mutant peptide contain both β- and α-sheets

Microscopic Insight into the Protein Denaturation Action of Urea and Its Methyl Derivatives

We employ site-specific, linear and nonlinear infrared spectroscopic techniques as well as fluorescence spectroscopy and molecular dynamics simulations to investigate the binding interactions of urea and three of its derivatives, methylurea, 1,3-dimethylurea, and tetramethylurea, with protein aromatic and polar side chains. We find that (1) urea methylation leads to preferential interactions between the cosolvent molecules and aromatic side chains with an affinity that increases with the number of methyl groups; (2) interactions with tetramethylurea cause significant dehydration of aromatic side chains and the effect is most pronounced for tryptophan; and (3) while neither urea nor tetramethylurea shows preferential accumulation around a polar side chain, the number of hydrogen-bond donors around this side chain is significantly decreased in the presence of tetramethylurea. Taken together, our findings suggest that these urea derivatives, especially tetramethylurea, can effectively disrupt hydrophobic interactions in proteins. Additionally, tetramethylurea can promote intramolecular hydrogen-bond formation and hence induce α-helix folding in peptides, as observed

Site-Specific Orientation of an α‑Helical Peptide Ovispirin‑1 from Isotope-Labeled SFG Spectroscopy

Sum-frequency generation (SFG) vibrational spectroscopy is often used to probe the backbone structures and orientations of polypeptides at surfaces. Using the ovispirin-1 polypeptide at the solid/liquid interface of polystyrene, we demonstrate for the first time that SFG can probe the polarization response of a single-isotope-labeled residue. To interpret the spectral intensities, we simulated the spectra using an excitonic Hamiltonian approach. We show that the polarization dependence of either the label or the unlabeled amide I band alone does not provide sufficient structural constraints to obtain both the tilt and the twist of the ovispirin helix at a solid/liquid interface, but that both can be determined from the polarization dependence of the complete spectrum. For ovispirin, the detailed analysis of the polarized SFG experimental data shows that the helix axis is tilted at roughly 138° from the surface normal, and the transition dipole of the isotope-labeled CO group is tilted at 23° from the surface normal, with the hydrophobic region facing the polystyrene surface. We further demonstrate that the Hamiltonian approach is able to address the coupling effect and the structural disorder. For comparison, we also collected the FTIR spectrum of ovispirin under similar conditions, which reveals the enhanced sensitivity of SFG for structural studies of single monolayer peptide surfaces. Our study provides insight into how structural and environmental effects appear in SFG spectra of the amide I band and establishes that SFG of isotope-labeled peptides will be a powerful technique for elucidating secondary structures with residue-by-residue resolution

Unveiling the Membrane-Binding Properties of N‑Terminal and C‑Terminal Regions of G Protein-Coupled Receptor Kinase 5 by Combined Optical Spectroscopies

G protein-coupled receptor kinase 5 (GRK5) is thought to associate with membranes in part via N- and C-terminal segments that are typically disordered in available high-resolution crystal structures. Herein we investigate the interactions of these regions with model cell membrane using combined sum frequency generation (SFG) vibrational spectroscopy and attenuated total reflectance–Fourier transform infrared (ATR-FTIR) spectroscopy. It was found that both regions associate with POPC lipid bilayers but adopt different structures when doing so: GRK5 residues 2–31 (GRK5_2–31) was in random coil whereas GRK5_546–565 was partially helical. When the subphase for the GRK5_2–31 peptide was changed to 40% TFE/60% 10 mM phosphate pH 7.4 buffer, a large change in the SFG amide I signal indicated that GRK5_2–31 became partially helical. By inspecting the membrane behavior of two different segments of GRK5_2–31, namely, GRK5_2–24 and GRK5_25–31, we found that residues 25–31 are responsible for membrane binding, whereas the helical character is imparted by residues 2–24. With SFG, we deduced that the orientation angle of the helical segment of GRK5_2–31 is 46 ± 1° relative to the surface normal in 40% TFE/60% 10 mM phosphate pH = 7.4 buffer but increases to 78 ± 11° with higher ionic strength. We also investigated the effect of PIP₂ in the model membrane and concluded that the POPC:PIP₂ (9:1) lipid bilayer did not change the behavior of either peptide compared to a pure POPC lipid bilayer. With ATR-FTIR, we also found that Ca²⁺·calmodulin is able to extract both peptides from the POPC lipid bilayer, consistent with the role of this protein in disrupting GRK5 interactions with the plasma membrane in cells

Gastric Cancer Staging with Dual Energy Spectral CT Imaging

<div><p>Purpose</p><p>To evaluate the clinical utility of dual energy spectral CT (DEsCT) in staging and characterizing gastric cancers.</p><p>Materials and Methods</p><p>96 patients suspected of gastric cancers underwent dual-phasic scans (arterial phase (AP) and portal venous phase (PP)) with DEsCT mode. Three types of images were reconstructed for analysis: conventional polychromatic images, material-decomposition images, and monochromatic image sets with photon energies from 40 to 140 keV. The polychromatic and monochromatic images were compared in TNM staging. The iodine concentrations in the lesions and lymph nodes were measured on the iodine-based material-decomposition images. These values were further normalized against that in aorta and the normalized iodine concentration (nIC) values were statistically compared. Results were correlated with pathological findings.</p><p>Results</p><p>The overall accuracies for T, N and M staging were (81.2%, 80.0%, and 98.9%) and (73.9%, 75.0%, and 98.9%) determined with the monochromatic images and the conventional kVp images, respectively. The improvement of the accuracy in N-staging using the keV images was statistically significant (p<0.05). The nIC values between the differentiated and undifferentiated carcinoma and between metastatic and non-metastatic lymph nodes were significantly different both in AP (p = 0.02, respectively) and PP (p = 0.01, respectively). Among metastatic lymph nodes, nIC of the signet-ring cell carcinoma were significantly different from the adenocarcinoma (p = 0.02) and mucinous adenocarcinoma (p = 0.01) in PP.</p><p>Conclusion</p><p>The monochromatic images obtained with DEsCT may be used to improve the N-staging accuracy. Quantitative iodine concentration measurements may be helpful for differentiating between differentiated and undifferentiated gastric carcinoma, and between metastatic and non-metastatic lymph nodes.</p></div

Dissecting the Ultrafast Stepwise Bidirectional Proton Relay in a Blue-Light Photoreceptor

Proton relays through H-bond networks are essential in realizing the functionality of protein machines such as in photosynthesis and photoreceptors. It has been challenging to dissect the rates and energetics of individual proton-transfer steps during the proton relay. Here, we have designed a proton rocking blue light using a flavin (BLUF) domain with the flavin mononucleotide (FMN)–glutamic acid (E)–tryptophan (W) triad and have resolved the four individual proton-transfer steps kinetically using ultrafast spectroscopy. We have found that after the photo-induced charge separation forming FMN·–/E-COOH/WH·+, the proton first rapidly jumps from the bridging E-COOH to FMN– (τfPT2 = 3.8 ps; KIE = 1.0), followed by a second proton transfer from WH·+ to E-COO– (τfPT1 = 336 ps; KIE = 2.6) which immediately rocks back to W· (τrPT1 = 85 ps; KIE = 6.7), followed by a proton return from FMNH· to E-COO– (τrPT2 = 34 ps; KIE = 3.3) with the final charge recombination between FMN·– and WH·+ to close the reaction cycle. Our results revisited the Grotthuss mechanism on the ultrafast timescale using the BLUF domain as a paradigm protein

Accuracies, sensitivities and specificities for N staging using kVp images (Group A) and optimal monochromatic images (Group B) with histological examination as the reference standard.

<p>Accuracies, sensitivities and specificities for N staging using kVp images (Group A) and optimal monochromatic images (Group B) with histological examination as the reference standard.</p

Same patient as Figure 3.

<p>Monochromatic image in portal phase demonstrated striation enhancement of blurring and wide reticular strands surrounding the outer border (arrow heads) of the tumor staged as T3 which was proved by histology.</p