Fourier-transform infrared spectroscopy for rapid screening and live-cell monitoring: application to nanotoxicology.

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. This alternative approach provides the ability to carry out rapid toxicity screenings and nondisruptive monitoring of live-cell cultures. We review the technical basis of the approach, the instrument configuration and interface with the biological media, the various effects that impact the data, subsequent data analysis and toxicity, and present some preliminary results on live-cell monitoring.

The effects of low-dose irradiation on inflammatory response proteins in a 3D reconstituted human skin tissue model.

Skin 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. To address this, we have utilized a reconstituted human skin tissue model (MatTek EpiDermFT™) and assessed changes in 23 cytokines, 24 and 48 h after treatment of skin with either 3 or 10 cGy low dose of radiation. Three cytokines, IFN-γ, IL-2, MIP-1α, were significantly altered in response to low-dose radiation. In contrast, seven cytokines were significantly altered in response to a high radiation dose of 200 cGy (IL-2, IL-10, IL-13, IFN-γ, MIP-1α, TNFα and VEGF) or the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (G-CSF, GM-CSF, IL-1α, IL-8, MIP-1α, MIP-1ß and RANTES). Additionally, radiation induced inflammation appears to have a distinct cytokine response relative to the nonradiation induced stressor, TPA. Overall, these results indicate that there are subtle changes in the inflammatory protein levels after exposure to low-dose radiation and this response is a subset of what is seen after a high dose in a human skin tissue model.

An altered mode of calcium coordination in methionine-oxidized calmodulin.

Oxidation 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. Changes in carboxylate ligands are consistent with the model we propose: an Asp at position 1 of the EF-loop experiences diminished hydrogen bonding with the polypeptide backbone, an Asp at position 3 forms a bidentate coordination of calcium, and an Asp at position 5 forms a pseudobridging coordination with a calcium-bound water molecule. The bidentate coordination of calcium by conserved glutamates is unaffected by oxidation. The observed changes in calcium ligation are discussed in terms of the placement of methionine side chains relative to the calcium-binding sites, suggesting that varying sensitivities of binding sites to oxidation may underlie the loss of CaM function upon oxidation.

A method for selective enrichment and analysis of nitrotyrosine-containing peptides in complex proteome samples.

Elevated 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. The procedure consists of the derivatization of nitrotyrosine into free sulfhydryl groups followed by high efficiency enrichment of sulfhydryl-containing peptides with thiopropyl sepharose beads. The derivatization process includes: (1) acetylation with acetic anhydride to block all primary amines, (2) reduction of nitrotyrosine to aminotyrosine, (3) derivatization of aminotyrosine with N-Succinimidyl S-Acetylthioacetate (SATA), and (4) deprotection of S-acetyl on SATA to form free sulfhydryl groups. The high specificity of this method is demonstrated by the contrasting percentage of nitrotyrosine-derivatized peptides in the identified tandem mass spectra between enriched and unenriched samples. Global analysis of unenriched in vitro nitrated human histone H1.2, bovine serum albumin (BSA), and mouse brain homogenate samples had 9%, 9%, and 5.9% of identified nitrotyrosine-containing peptides, while the enriched samples had 91% , 62%, and 35%, respectively. Duplicate LC-MS/MS analyses of the enriched mouse brain homogenate identified 150 unique nitrated peptides covering 102 proteins with an estimated 3.3% false discovery rate.

Endogenously nitrated proteins in mouse brain: links to neurodegenerative disease.

Increased 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. More than half of the nitrated proteins that have been identified are involved in Parkinson's disease, Alzheimer's disease, or other neurodegenerative disorders. Similarly, nitrotyrosine immunoblots of whole brain homogenates show that treatment of mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), an experimental model of Parkinson's disease, induces an increased level of nitration of the same protein bands observed to be nitrated in brains of untreated animals. Comparing sequences and available high-resolution structures around nitrated tyrosines with those of unmodified sites indicates a preference of nitration in vivo for surface accessible tyrosines in loops, a characteristic consistent with peroxynitrite-induced tyrosine modification. In addition, most sequences contain cysteines or methionines proximal to nitrotyrosines, contrary to suggestions that these amino acid side chains prevent tyrosine nitration. More striking is the presence of a positively charged moiety near the sites of nitration, which is not observed for non-nitrated tyrosines. Together, these observations suggest a predictive tool of functionally important sites of nitration and that cellular nitrating conditions play a role in neurodegenerative changes in the brain.

Tertiary structural rearrangements upon oxidation of Methionine145 in calmodulin promotes targeted proteasomal degradation.

The 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. To confirm the specificity of CaMox degradation and to identify the structural signals underlying the preferential recognition and degradation by the proteasome/Hsp90, we have investigated how the oxidation of individual methionines affect the degradation of CaM using mutants in which all but selected methionines in CaM were substituted with leucines. Substitution of all methionines with leucines except Met144 and Met145 has no detectable effect on the structure of CaM, permitting a determination of how site-specific substitutions and the oxidation of Met144 and Met145 affects the recognition and degradation of CaM by the proteasome/Hsp90. Comparable rates of degradation are observed upon the selective oxidation of Met144 and Met145 in CaM-L7 relative to that observed upon oxidation of all nine methionines in wild-type CaM. Substitution of leucines for either Met144 or Met145 promotes a limited recognition and degradation by the proteasome that correlates with decreases in the helical content of CaM. The specific oxidation of Met144 has little effect on rates of proteolytic degradation by the proteasome/Hsp90 or the structure of CaM. In contrast, the specific oxidation of Met145 results in both large increases in the rate of degradation by the proteasome/Hsp90 and significant circular dichroic spectral shape changes that are indicative of changes in tertiary rather than secondary structure. Thus, tertiary structural changes resulting from the site-specific oxidation of a single methionine (i.e., Met145) promote the degradation of CaM by the proteasome/Hsp90, suggesting a mechanism to regulate cellular metabolism through the targeted modulation of CaM abundance in response to oxidative stress.

Essential role for Pro21 in phospholamban for optimal inhibition of the Ca-ATPase.

We 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. In agreement with prior measurements in cardiac SR microsomes, the calcium concentration associated with half-maximal activation (Ca(1/2)) of the Ca-ATPase, 290 +/- 10 nM, is shifted to 580 +/- 20 nM when co-reconstituted with PLB(C) (Pro21) as a result of a reduction in the cooperativity associated with the calcium-dependent structural transition. Kinetic simulations indicate that PLB(C) association with the Ca-ATPase results in a 75% reduction in the equilibrium constant associated with the formation of the second high-affinity calcium binding site. In comparison, there is a 43% reduction in KCa(1/2) upon reconstitution of the Ca-ATPase with P21A-PLB(C), which can be simulated by decreasing the equilibrium constant associated with the calcium-dependent structural activation by 50%. The diminished inhibitory action of P21A-PLB(C) is associated with alterations in the structure of the hinge element, as evidenced by the diminished solvent accessibility of FMal relative to the native structure. Likewise, increases in the alpha-helical content and decreases in the mobility of the carboxyl-terminal domain of P21A-PLB(C) are observed using circular dichroism and fluorescence spectroscopy. Collectively, these results indicate that the overall dimensions of the carboxyl-terminal domain of PLB are increased through a stabilization of secondary structural elements upon mutation in P21A-PLB(C) that result in a reduction in the ability of the amino-terminal cytosolic portion of PLB to productively inhibit the Ca-ATPase. Further, these results suggest that the unstructured characteristics of the flexible hinge region in PLB are critical for optimal inhibitory interactions with the Ca-ATPase and suggest its role as a conformational switch.

Role for bound water and CH-pi aromatic interactions in photosynthetic electron transfer.

Photosystem 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.; Xu, W.; Martinsson, P.; Chitnis, P. R.; Savikhin, S. Biophys. J. 2004, 86, 3121. Kim, S.; Sacksteder, C. A.; Bixby, K. A.; Barry, B. A. Biochemistry 2001, 40, 15384). To investigate the details of electron transfer and relaxation events in PSI, we have employed several experimental approaches. First, we report a pH-dependent viscosity effect on P700+ reduction; this result suggests a role for proton transfer in the PSI electron-transfer reactions. Second, we find that changes in hydration alter the rate of P700+ reduction and the interactions of P700 with the protein environment. This result suggests a role for bound water in electron transfer to P700+. Third, we present evidence that deuteration of the tyrosine aromatic side chain perturbs the vibrational spectrum, associated with P700+ reduction. We attribute this result to a linkage between CH-pi interactions and electron transfer to P700+.

Redox-active tyrosine residues: role for the peptide bond in electron transfer.

Photosystem 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. PSII data were acquired on control samples, as well as samples in which tyrosine was 2H4 (ring)-labeled. Comparison to model compound data, acquired both from tyrosinate and its 2H4 isotopomer, was performed. The PSII FT-IR spectrum exhibited vibrational bands that are assignable to imide and amide vibrational modes. In previous work, we have shown that oxidation of tyrosinate perturbs the terminal amino group of tyrosinate (Ayala, I.; Range, K.; York, D.; Barry, B. A. J. Am. Chem. Soc. 2002, 124, 5496-5505). Density functional calculations on tyrosinate supported the interpretation that the perturbation is due to spin delocalization onto the amino group. In tyrosine-containing dipeptides, perturbations of the peptide bond were observed. Therefore, the imide and amide perturbations observed here are attributed to spin delocalization into the peptide bond in PSII. Migration of the electron hole in PSII may be consistent with peptide bond involvement in tyrosyl radical-based electron-transfer reactions.