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- ItemDetection of Protein Glycosylation Using Tip-Enhanced Raman Scattering(Columbus, Ohio : American Chemical Society, 2016) Cowcher, David P.; Deckert-Gaudig, Tanja; Brewster, Victoria L.; Ashton, Lorna; Deckert, Volker; Goodacre, RoystonThe correct glycosylation of biopharmaceutical glycoproteins and their formulations is essential for them to have the desired therapeutic effect on the patient. It has recently been shown that Raman spectroscopy can be used to quantify the proportion of glycosylated protein from mixtures of native and glycosylated forms of bovine pancreatic ribonuclease (RNase). Here we show the first steps toward not only the detection of glycosylation status but the characterization of glycans themselves from just a few protein molecules at a time using tip-enhanced Raman scattering (TERS). While this technique generates complex data that are very dependent on the protein orientation, with the careful development of combined data preprocessing, univariate and multivariate analysis techniques, we have shown that we can distinguish between the native and glycosylated forms of RNase. Many glycoproteins contain populations of subtly different glycoforms; therefore, with stricter orientation control, we believe this has the potential to lead to further glycan characterization using TERS, which would have use in biopharmaceutical synthesis and formulation research.
- ItemHigh-throughput screening Raman microspectroscopy for assessment of drug-induced changes in diatom cells(Cambridge : Royal Society of Chemistry, 2019) Rüger J.; Mondol A.S.; Schie I.W.; Popp J.; Krafft C.High-throughput screening Raman spectroscopy (HTS-RS) with automated localization algorithms offers unsurpassed speed and sensitivity to investigate the effect of dithiothreitol on the diatom Phaedactylum tricornutum. The HTS-RS capability that was demonstrated for this model system can be transferred to unmet analytical applications such as kinetic in vivo studies of microalgal assemblages. © 2019 The Royal Society of Chemistry.
- ItemFemtosecond stimulated Raman spectroscopy of the cyclobutane thymine dimer repair mechanism: A computational study(Washington, DC : American Chemical Society, 2014) Ando, H.; Fingerhut, B.P.; Dorfman, K.E.; Biggs, J.D.; Mukamel, S.Cyclobutane thymine dimer, one of the major lesions in DNA formed by exposure to UV sunlight, is repaired in a photoreactivation process, which is essential to maintain life. The molecular mechanism of the central step, i.e., intradimer C-C bond splitting, still remains an open question. In a simulation study, we demonstrate how the time evolution of characteristic marker bands (C=O and C=C/C-C stretch vibrations) of cyclobutane thymine dimer and thymine dinucleotide radical anion, thymidylyl(3′→5′)-thymidine, can be directly probed with femtosecond stimulated Raman spectroscopy (FSRS). We construct a DFT(M05-2X) potential energy surface with two minor barriers for the intradimer C5-C′5 splitting and a main barrier for the C6-C′6 splitting, and identify the appearance of two C5=C6 stretch vibrations due to the C6-C′6 splitting as a spectroscopic signature of the underlying bond splitting mechanism. The sequential mechanism shows only absorptive features in the simulated FSRS signals, whereas the fast concerted mechanism shows characteristic dispersive line shapes. (Figure Presented).
- ItemCharge isomers of myelin basic protein: Structure and interactions with membranes, nucleotide analogues, and calmodulin(San Francisco, CA : Public Library of Science, 2011) Wang, C.; Neugebauer, U.; Bürck, J.; Myllykoski, M.; Baumgärtel, P.; Popp, J.; Kursula, P.As an essential structural protein required for tight compaction of the central nervous system myelin sheath, myelin basic protein (MBP) is one of the candidate autoantigens of the human inflammatory demyelinating disease multiple sclerosis, which is characterized by the active degradation of the myelin sheath. In this work, recombinant murine analogues of the natural C1 and C8 charge components (rmC1 and rmC8), two isoforms of the classic 18.5-kDa MBP, were used as model proteins to get insights into the structure and function of the charge isomers. Various biochemical and biophysical methods such as size exclusion chromatography, calorimetry, surface plasmon resonance, small angle X-ray and neutron scattering, Raman and fluorescence spectroscopy, and conventional as well as synchrotron radiation circular dichroism were used to investigate differences between these two isoforms, both from the structural point of view, and regarding interactions with ligands, including calmodulin (CaM), various detergents, nucleotide analogues, and lipids. Overall, our results provide further proof that rmC8 is deficient both in structure and especially in function, when compared to rmC1. While the CaM binding properties of the two forms are very similar, their interactions with membrane mimics are different. CaM can be used to remove MBP from immobilized lipid monolayers made of synthetic lipids - a phenomenon, which may be of relevance for MBP function and its regulation. Furthermore, using fluorescently labelled nucleotides, we observed binding of ATP and GTP, but not AMP, by MBP; the binding of nucleoside triphosphates was inhibited by the presence of CaM. Together, our results provide important further data on the interactions between MBP and its ligands, and on the differences in the structure and function between MBP charge isomers.
- ItemMicrobial respiration and natural attenuation of benzene contaminated soils investigated by cavity enhanced Raman multi-gas spectroscopy(Cambridge : Soc., 2015) Jochum, Tobias; Michalzik, Beate; Bachmann, Anne; Popp, Jürgen; Frosch, TorstenSoil and groundwater contamination with benzene can cause serious environmental damage. However, many soil microorganisms are capable to adapt and are known to strongly control the fate of organic contamination. Innovative cavity enhanced Raman multi-gas spectroscopy (CERS) was applied to investigate the short-term response of the soil micro-flora to sudden surface contamination with benzene regarding the temporal variations of gas products and their exchange rates with the adjacent atmosphere. 13C-labeled benzene was spiked on a silty-loamy soil column in order to track and separate the changes in heterotrophic soil respiration – involving 12CO2 and O2 – from the natural attenuation process of benzene degradation to ultimately form 13CO2. The respiratory quotient (RQ) decreased from a value 0.98 to 0.46 directly after the spiking and increased again within 33 hours to a value of 0.72. This coincided with the maximum 13CO2 concentration rate (0.63 μmol m−2 s−1), indicating the highest benzene degradation at 33 hours after the spiking event. The diffusion of benzene in the headspace and the biodegradation into 13CO2 were simultaneously monitored and 12 days after the benzene spiking no measurable degradation was detected anymore. The RQ finally returned to a value of 0.96 demonstrating the reestablished aerobic respiration.
- ItemOnline investigation of respiratory quotients in Pinus sylvestris and Picea abies during drought and shading by means of cavity-enhanced Raman multi-gas spectrometry(Cambridge : Soc., 2015) Hanf, Stefan; Fischer, Sarah; Hartmann, Henrik; Keiner, Robert; Trumbore, Susan; Popp, Jürgen; Frosch, TorstenPhotosynthesis and respiration are major components of the plant carbon balance. During stress, like drought, carbohydrate supply from photosynthesis is reduced and the Krebs cycle respiration must be fueled with other stored carbon compounds. However, the dynamics of storage use are still unknown. The respiratory quotient (RQ, CO2 released per O2 consumed during respiration) is an excellent indicator of the nature of the respiration substrate. In plant science, however, online RQ measurements have been challenging or even impossible so far due to very small gas exchange fluxes during respiration. Here we apply cavity-enhanced multi-gas Raman spectrometry (CERS) for online in situ RQ measurements in drought-tolerant pine (Pinus sylvestris [L.]) and drought-intolerant spruce (Picea abies [L. H. Karst]). Two different treatments, drought and shading, were applied to reduce photosynthesis and force dependency on stored substrates. Changes in respiration rates and RQ values were continuously monitored over periods of several days with low levels of variance. The results show that both species switched from COH-dominated respiration (RQ = 1.0) to a mixture of substrates during shading (RQ = 0.77–0.81), while during drought only pine did so (RQ = 0.75). The gas phase measurements were complemented by concentration measurements of non-structural carbohydrates and lipids. These first results suggest a physiological explanation for greater drought tolerance in pine. CERS was proven as powerful technique for non-consumptive and precise real-time monitoring of respiration rates and respirational quotients for the investigation of plant metabolism under drought stress conditions that are predicted to increase with future climate change.
- ItemSpatially resolved spectroscopic differentiation of hydrophilic and hydrophobic domains on individual insulin amyloid fibrils([London] : Macmillan Publishers Limited, part of Springer Nature, 2016) Deckert-Gaudig, Tanja; Kurouski, Dmitry; Hedegaard, Martin A. B.; Singh, Pushkar; Lednev, Igor K.; Deckert, VolkerThe formation of insoluble β-sheet-rich protein structures known as amyloid fibrils is associated with numerous neurodegenerative diseases, such as Alzheimer’s and Parkinson’s disease. A detailed understanding of the molecular structure of the fibril surface is of interest as the first contact with the physiological environment in vivo and plays a decisive role in biological activity and associated toxicity. Recent studies reveal that the inherent sensitivity and specificity of tip-enhanced Raman scattering (TERS) renders this technique a compelling method for fibril surface analysis at the single-particle level. Here, the reproducibility of TERS is demonstrated, indicating its relevance for detecting molecular variations. Consequently, individual fibrils are systematically investigated at nanometer spatial resolution. Spectral parameters were obtained by band-fitting, particularly focusing on the identification of the secondary structure via the amide III band and the differentiation of hydrophobic and hydrophilic domains on the surface. In addition multivariate data analysis, specifically the N-FINDR procedure, was employed to generate structure-specific maps. The ability of TERS to localize specific structural domains on fibril surfaces shows promise to the development of new fibril dissection strategies and can be generally applied to any (bio)chemical surface when structural variations at the nanometer level are of interest.
- ItemSingle cell analysis in native tissue: Quantification of the retinoid content of hepatic stellate cells([London] : Macmillan Publishers Limited, part of Springer Nature, 2016) Galler, Kerstin; Requardt, Robert Pascal; Glaser, Uwe; Markwart, Robby; Bocklitz, Thomas; Bauer, Michael; Popp, Jürgen; Neugebauer, UteHepatic stellate cells (HSCs) are retinoid storing cells in the liver: The retinoid content of those cells changes depending on nutrition and stress level. There are also differences with regard to a HSC’s anatomical position in the liver. Up to now, retinoid levels were only accessible from bulk measurements of tissue homogenates or cell extracts. Unfortunately, they do not account for the intercellular variability. Herein, Raman spectroscopy relying on excitation by the minimally destructive wavelength 785 nm is introduced for the assessment of the retinoid state of single HSCs in freshly isolated, unprocessed murine liver lobes. A quantitative estimation of the cellular retinoid content is derived. Implications of the retinoid content on hepatic health state are reported. The Raman-based results are integrated with histological assessments of the tissue samples. This spectroscopic approach enables single cell analysis regarding an important cellular feature in unharmed tissue.