A significant challenge to realize the full potential of nanotechnology for therapeutic and diagnostic applications is to understand and evaluate how live cells interact with an external stimulus, such as a nanosized particle, and the toxicity and broad risk associated with these stimuli. It is difficult to capture the complexity and dynamics of these interactions by following omics-based approaches exclusively, which can be expensive and time-consuming. Attenuated total reflectance-Fourier transform infrared spectroscopy is well suited to provide noninvasive live-cell monitoring of cellular responses to potentially toxic nanosized particles or other stimuli.
View Article and Find Full Text PDFSkin responses to moderate and high doses of ionizing radiation include the induction of DNA repair, apoptosis and stress response pathways. Additionally, numerous studies indicate that radiation exposure leads to inflammatory responses in skin cells and tissue. However, the inflammatory response of skin tissue to low-dose radiation (≤10 cGy) is poorly understood.
View Article and Find Full Text PDFOxidation of methionine residues in calmodulin (CaM) lowers the affinity for calcium and results in an inability to activate target proteins fully. To evaluate the structural consequences of CaM oxidation, we used infrared difference spectroscopy to identify oxidation-dependent effects on protein conformation and calcium liganding. Oxidation-induced changes include an increase in hydration of alpha-helices, as indicated in the downshift of the amide I' band of both apo-CaM and Ca(2+)-CaM, and a modification of calcium liganding by carboxylate side chains, reflected in antisymmetric carboxylate band shifts.
View Article and Find Full Text PDFElevated levels of protein tyrosine nitration have been found in various neurodegenerative diseases and age-related pathologies. Until recently, however, the lack of an efficient enrichment method has prevented the analysis of this important low-level protein modification. We have developed a method that specifically enriches nitrotyrosine-containing peptides so that both nitrotyrosine peptides and specific nitration sites can be unambiguously identified with LC-MS/MS.
View Article and Find Full Text PDFIncreased abundance of nitrotyrosine modifications of proteins have been documented in multiple pathologies in a variety of tissue types and play a role in the redox regulation of normal metabolism. To identify proteins sensitive to nitrating conditions in vivo, a comprehensive proteomic data set identifying 7792 proteins from a whole mouse brain, generated by LC/LC-MS/MS analyses, was used to identify nitrated proteins. This analysis resulted in the identification of 31 unique nitrotyrosine sites within 29 different proteins.
View Article and Find Full Text PDFThe selectivity underlying the recognition of oxidized calmodulin (CaM) by the 20S proteasome in complex with Hsp90 was identified using mass spectrometry. We find that degradation of oxidized CaM (CaMox) occurs in a multistep process, which involves an initial cleavage that releases a large N-terminal fragment (A1-F92) as well as multiple smaller carboxyl-terminus peptides ranging from 17 to 26 amino acids in length. These latter small peptides are enriched in methionine sulfoxides (MetO), suggesting a preferential degradation around MetO within the carboxyl-terminal domain.
View Article and Find Full Text PDFWe have investigated the functional role of the flexible hinge region centered near the sequence TIEMP(21), which connects the N-terminal cytosolic and C-terminal membrane-spanning helical domains of phospholamban (PLB). Specifically, we ask if the conformation of this region is important to attain optimal inhibitory interactions with the Ca-ATPase. A genetically engineered PLB mutant was constructed in which Pro(21) was mutated to an alanine (P21A-PLB(C)); in this construct, all three transmembrane cysteines were substituted with alanines to stabilize the monomeric form of PLB, and a unique cysteine was introduced at position 24 near the hinge element (A24C), permitting the site-specific attachment of fluorescein-5-maleimide (FMal) to monitor structure changes.
View Article and Find Full Text PDFWe introduce a new, non-invasive technique to measure linear electron transfer in intact leaves under steady-state illumination. Dark-interval relaxation kinetic or 'DIRK' analysis is based on measurements of the initial rates of relaxation of steady-state absorbance signals upon a rapid light-dark transition. We show that estimates of electron flux by DIRK analysis of absorbance signals, reflecting redox changes in the photosynthetic electron transfer chain, can yield quantitative information about photosynthetic flux when the light-dependent partitioning of electrons among redox components of the electron transfer chain are considered.
View Article and Find Full Text PDFKinetically-resolved absorbance measurements during extended, or steady-state illumination are typically hindered by large, light-induced changes in the light-scattering properties of the material. In this work, a new type of portable spectrophotometer, the Non-Focusing Optical Spectrophotometer (NoFOSpec), is introduced, which reduces interference from light-scattering changes and is in a form suitable for fieldwork. The instrument employs a non-focusing optical component, called a compound parabolic concentrator (CPC), to simultaneously concentrate and homogeneously diffuse measuring and actinic light (from light-emitting diode sources) onto the leaf sample.
View Article and Find Full Text PDFPhotosystem I (PSI) is one of two photosynthetic reaction centers present in plants, algae, and cyanobacteria and catalyzes the reduction of ferredoxin and the oxidation of cytochrome c or plastocyanin. The PSI primary chlorophyll donor, which is oxidized in the primary electron-transfer events, is a heterodimer of chl a and a' called P700. It has been suggested that protein relaxation accompanies light-induced electron transfer in this reaction center (Dashdorj, N.
View Article and Find Full Text PDFPhotosystem II (PSII) catalyzes the light-driven oxidation of water and reduction of plastoquinone. In PSII, redox-active tyrosine Z conducts electrons between the primary chlorophyll donor and the manganese cluster, which is the catalytic site. In this report, difference FT-IR spectroscopy is used to show that oxidation of redox-active tyrosine Z causes perturbations of the peptide bond.
View Article and Find Full Text PDFIn oxygenic photosynthesis, photosystem I (PSI) conducts light-driven electron transfer from plastocyanin to ferredoxin. The reactions are initiated when the primary chlorophyll donor, P(700), is photooxidized. P(700) is a chlorophyll dimer ligated by the core subunits psaA and psaB.
View Article and Find Full Text PDFChloroplasts in bundle sheath cells (BSC) of maize perform photosystem I (PSI)-mediated production of ATP. In this study, the participation of ascorbate (Asc) as an electron donor to PSI in light-induced electron transport in isolated maize BSC was demonstrated. It was found that Asc, at physiological concentrations, rapidly reduced photooxidized reaction center chlorophyll of PSI (P700).
View Article and Find Full Text PDFThe observed levels of Delta G(ATP) in chloroplasts, as well as the activation behavior of the CF(1)CF(0)-ATP synthase, suggest a minimum transthylakoid proton motive force (pmf) equivalent to a Delta pH of approximately 2.5 units. If, as is commonly believed, all transthylakoid pmf is stored as Delta pH, this would indicate a lumen pH of less than approximately 5.
View Article and Find Full Text PDFA noninvasive technique is introduced with which relative proton to electron stoichiometries (H(+)/e(-) ratios) for photosynthetic electron transfer can be obtained from leaves of living plants under steady-state illumination. Both electron and proton transfer fluxes were estimated by a modification of our previously reported dark-interval relaxation kinetics (DIRK) analysis, in which processes that occur upon rapid shuttering of the actinic light are analyzed. Rates of turnover of linear electron transfer through the cytochrome (cyt) b(6)f complex were estimated by measuring the DIRK signals associated with reduction of cyt f and P(700).
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