Prodan Fluorescence Mimics the GroEL Folding Cycle

The non-covalent fluorescent probe 6-propionyl-2-(dimethylamino) naphthalene sulfonate (prodan) binds to hydrophobic surfaces exposed on the surface of GroEL. Under identical experimental conditions free prodan exhibits a green emission peak of intensity 390,000 cps at 520 nm. However prodan bound to GroEL, GroEL–ATP, and GroEL–ATP–GroES shows emission peaks of intensities 500,000, 540,000, and 480,000 cps at 515, 512 and 515 nm, respectively, thus mimicing the way hydrophobic surfaces on GroEL become exposed during the folding cycle. Other hydrophobic probes like bis-ANS and dansyl lysine were unable to detect the minor changes in hydrophobic exposure of GroEL after it binds to ATP, although they were able to detect hydrophobic exposure in GroEL itself.     

Quantitative Protein Topography Analysis and High-Resolution Structure Prediction Using Hydroxyl Radical Labeling and Tandem-Ion Mass Spectrometry (MS)

Hydroxyl radical footprinting based MS for protein structure assessment has the goal of understanding ligand induced conformational changes and macromolecular interactions, for example, protein tertiary and quaternary structure, but the structural resolution provided by typical peptide-level quantification is limiting. In this work, we present experimental strategies using tandem-MS fragmentation to increase the spatial resolution of the technique to the single residue level to provide a high precision tool for molecular biophysics research. Overall, in this study we demonstrated an eightfold increase in structural resolution compared with peptide level assessments. In addition, to provide a quantitative analysis of residue based solvent accessibility and protein topography as a basis for high-resolution structure prediction; we illustrate strategies of data transformation using the relative reactivity of side chains as a normalization strategy and predict side-chain surface area from the footprinting data. We tested the methods by examination of Ca⁺²-calmodulin showing highly significant correlations between surface area and side-chain contact predictions for individual side chains and the crystal structure. Tandem ion based hydroxyl radical footprinting-MS provides quantitative high-resolution protein topology information in solution that can fill existing gaps in structure determination for large proteins and macromolecular complexes.Hydroxyl radical footprinting (HRF)¹ is valuable for assessing the structure of macromolecules. Single nucleotide resolution data enabled by the similar reactivity of the OH radical with each and every backbone position has helped solve important problems in the nucleic acids field, such as understanding RNA folding and ribosome assembly (1–5). Applications of HRF to probe protein structure are a subset of a family of structural MS approaches, including the use of reversible deuterium labeling or irreversible covalent labeling, including labeling with OH radicals (6–13). Hydrogen-deuterium exchange MS (HDX-MS) is particularly suited to measure secondary and tertiary structure stability through backbone exchange, whereas HRF-MS has been effective at measuring the relative solvent accessibility of specific amino acid side chains mediated by intramolecular tertiary and intermolecular quaternary structure interactions. Hydroxyl radicals can be generated by a variety of methods in each case the chemistry has been shown to be quite similar and the radicals react with side chains of surface residues resulting in well characterized oxidation products (7, 10, 11). As up to 18 side chains are potential probes, the overall protein coverage and resolution of the method is theoretically high.Both HDX-MS and HRF-MS utilize a “bottom-up” proteomics approach where proteins are digested to peptide states after labeling, and mass shifts of the resultant peptides are read-out to pinpoint sites of conformational change. Although this usually can provide 90% or more coverage across the entire protein length, in fact the structural resolution is limited as the size of the peptide fragments and the data report the average behavior of the individual residues across the entire peptide, which are typically in the range of five–20 residues (14). MS2 based quantification is in principle a general solution to the problem of increasing structural resolution, and has been attempted for HDX-MS, but the scrambling of the labels in the gas phase has been difficult to overcome using collision induced dissociation (15, 16). Alternative approaches for HDX-MS site localization, like electron transfer dissociation to achieve single residue resolution have potential promise but are typically limited to larger peptides that can access higher charge states easily (17, 18). MS2 strategies to enhance the resolution for covalent labeling experiments have been attempted with some success, as scrambling is not a limitation in covalent labeling experiments (7, 19–21). On the other hand, MS1 based strategies to enhance structural resolution for both HDX and covalent labeling approaches using overlapping protease fragments are also a promising route to providing subpeptide resolution in many cases (7, 20–27).In this work, we present a coupled set of high-throughput experimental and computational approaches to extend previous MS2 based HRF-MS strategies and provide a quantitative topographical structure assessment for proteins at the individual side chain level. The combined approach permits quantification of modifications through examination of a tandem-ion based ladder of peptide fragments and combining the ion abundances from both MS1 and MS2 quantification. The high-resolution information is transformed using the knowledge of the relative reactivity of side chains to predict side-chain surface area for the structurally well-characterized Ca²⁺ bound form of Calmodulin (CaM). In addition, we explored a statistical approach using random forest regression methods to predict solvent accessible surface area at the residue level. Overall, these studies provide a novel approach to provide high-resolution single-residue surface accessibility data with at least eightfold higher spatial resolution than peptide based measures for accurate protein topography predictions.     

Synthetic, immunological and structural studies on repeat unit peptides of Plasmodium falciparum antigens

Using solid phase methodology, we have synthesized five peptides (16-18 residues long) corresponding to repeat sequences of four antigens of a human malarial parasite, Plasmodium falciparum. Three of these antigens (RESA, FIRA, and ABRA) are found in the asexual blood-stages of the parasite, while the remaining one (CSP) is found in the sporozoites. The synthetic peptides, conjugated to bovine serum albumin, elicited high levels of antibodies in rabbits, and these antibodies were found to cross-react with the heterologous peptides. The degree of cross-reactivity, as estimated in an ELISA, was quite remarkable among all the peptides. The peptide corresponding to the RESA tetrapeptide repeat was found to be the most immunogenic and highly cross-reactive. For this reason this tetrapeptide repeat unit, peptide 1, may be a suitable candidate for inclusion in a multiple epitope polypeptide vaccine design. Conformational studies using circular dichroism spectroscopy show that these peptides have similar conformational characteristics with a common feature of approximately 30% and approximately 50% helical content water and TFE respectively. Theoretical predictions regarding conformation using the Chou-Fasman method have also been presented.     

Plasmodesmata the Nano Bridges in Plant Cell: Are the Answer for All the Developmental Processes?

Russian Journal of Plant Physiology - Plants establish tiny, cellular signaling pathways playing a unique coordination role in growth and development. Plasmodesmata are playing a central role in...     

Association of interleukin-18 genotypes (−607C > A) and (−137 G > C) with the hepatitis B virus disease progression to hepatocellular carcinoma

Chronic infection with HBV has been reported to be associated with the development of HCC. The inflammation mounted by cytokine-mediated immune system plays an important role in the pathogenesis of HBV-associated HCC. IL-18 is a pro-inflammatory cytokine whose role in the development of HBV-associated chronic to malignant disease state has not been much studied. The present study was conceived to determine the role of genetic polymorphisms in IL-18, serum levels of IL-18, and expression level of its signal transducers in the HBV disease progression. A total of 403 subjects were enrolled for this study including 102 healthy subjects and 301 patients with HBV infection in different diseased categories. Polymorphism was determined using PCR–RFLP. Genotypic distributions between the groups were compared using odd’s ratio and 95% CI were calculated to express the relative risk. Circulating IL-18 levels were determined by ELISA. Expression levels of pSTAT-1 and pNF?B was determined by western blotting. In case of IL-18(??607C?>?A), the heterozygous genotype (CA) was found to be a protective factor while in case of IL-18(??137G?>?C) the heterozygous genotype (GC) acted as a risk factor for disease progression from HBV to HCC. Moreover, serum IL-18 levels were significantly increased during HBV disease progression to HCC as compared to controls. Also the levels of activated signal transducers (pSTAT-1 and pNF-κB) of IL-18 in stimulated PBMCs were significantly increased during HBV to HCC disease progression. These findings suggest that IL-18 has the potential to act as a biomarker of HBV-related disease progression to HCC.