Mobility of free radicals connected with serum albumins at 30-230 K

This paper presents experimental results of inner dynamics study of human serum albumin by a series of physical methods: a) the method of radical pairs recombination based on measuring of recombination rate of radical pairs included to surface layers of a protein globule; b) the method of spin labels with saturation transfer technique. The complex of methods used in this work permits to measure the mobility of physical labels and environment matrix in a wide correlation frequency range 10(-4)-5.10(10) s-1 in 30-230 K temperature range. The analysis of the data obtained allows to make conclusions on the molecular mobility in surface layers of a protein globule under different conditions.     

Temperature-induced transitions of function and structure in sarcoplasmic reticulum membranes

A transition in the temperature dependences of Ca²⁺ accumulation and ATPase activity occurs at 20 ° C in Sarcoplasmic reticulum membranes. The transition is characterized by an abrupt change in the activation energies for the cation transport process and the associated enzyme activities. The difference in activation energies below and above 20 °C appears to be due to changes in the entropy of activation rather than in the free energy of activation. Also, the temperature dependences of spectral parameters of lipophilic spin-labeled probes and protein-bound spin labels exhibit different behaviors on either side of this temperature. Above 20 °C the lipid matrix probed by the labels exhibits a large increase in molecular motion and a decrease in the apparent ordering of lipid alkyl chains. In addition, labels covalently bound to enzymic reactive sites indicate that the motion of protein side-chains is sensitive to this transition. The results are consistent with an order-disorder transition involving the lipid alkyl chains of the Sarcoplasmic membrane, and with a model in which molecular motion, Ca²⁺ transport and enzyme activity are limited by local viscosity of hydrophobic regions at temperatures below the transition.Another modification of the Sarcoplasmic reticulum membrane occurs between 37 and 40 °C. It appears that at this temperature the processes governing Ca²⁺ accumulation and ATPase activity are uncoupled, and Ca²⁺ accumulation is inhibited, while ATPase activity and passive Ca²⁺ efflux proceed at rapid rates. Parallel transitions of spectroscopic parameters originating from spin labels, covalently bound to the Sarcoplasmic reticulum ATPase, indicate that the uncoupling is due to a thermally-induced protein conformational change.     

Fluorescence labels as sensors for oxygen binding of arthropod hemocyanins

The molecular basis of high cooperativity in multi-subunit proteins is still unknown in most cases. Oxygen binding by multi-subunit hemocyanins produces two intrinsic spectroscopic signals which are, however, either limited to the UV or are very weak. Here we demonstrate that fluorescence labels emitting in the visible can be used as sensors for cooperative oxygen binding of hemocyanins. Fluorescence resonance energy transfer to the oxygenated active sites quenches the emission of the labels by roughly 50% upon oxygenation of the protein. The labels give strong and photo-stable emission, allowing imaging of single hemocyanin molecules. Therefore, this study opens up a new perspective for investigating the molecular basis of cooperative oxygen binding at the single-molecule level. In addition, another novel application is provided by these labels, i.e., the investigation of the influence of effectors by recording simultaneously the binding of oxygen in the visible and of effectors in the UV.     

Cell-cycle-dependent changes in labelling of specific phosphoproteins by the monoclonal antibody MPM-2 in plant cells

The MPM-2 antibody, which recognizes a mitosis-specific phosphorylated epitope, has been used to study cell-cycle-related proteins in partially synchronized cell suspension cultures and root meristem cells. Immunofluorescence revealed that the epitope recognized by MPM-2 is located in the nucleus in interphase cells. In mitotic cells, MPM-2 labels the prophase nucleus, the spindle and some cytoplasmic components. The relative amount of the epitope changes significantly during the cell cycle. Labelling is lowest in G1 and S-phase cells and increases 2–3-fold during G2. Prophase and metaphase show four to five times the labelling of G1 cells. Labelling decreases rapidly after metaphase and is at a very low level by telophase. One- (1-D) and two-dimensional (2-D) immunoblots showed that MPM-2 labels a family of phosphorylated proteins. The labelling shows significant cell cycle dependence. Subfractionation shows at least one of these proteins is a component of the detergent-insoluble cytoskeleton cell fraction. This component is resolved on 2-D immunoblots to two to three spots of slightly different isoelectric point, possibly charge isomers, at a relative molecular mass of approximately 65 kDa. The same spots are labelled by IFA, an antibody against intermediate filament proteins. Another three of the spots at lower relative molecular mass are labelled on 2-D immunoblots of the nuclear matrix fraction.     

Changes in the restriction of molecular rotational diffusion of water-soluble spin labels during fatty acid starvation of yeast.

Yeast mutants lacking fatty acid synthetase activity (fas-) die when deprived of saturated fatty acid under conditions which are otherwise growth-supporting. The spin label technique is used to show that restriction of molecular rotational diffusion of spin label molecules dissolved in aqueous zones increases several fold under conditions of fatty acid starvation while the apparent physical state of cellular hydrocarbon zones remains essentially unchanged. We focus attention on the cellular aqueous interior as the potential site of alteration under selective starvation conditions. Correspondences exist between restriction of molecular motion of water soluble spin labels dissolved in the cell and loss of cell viability. The correspondences to changes in the molecular motion of hydrocarbon soluble spin labels are much less or are not detectable.     

Intrinsic proteins of the intestinal microvillus membrane. Iodonaphythylazide labeling studies

Isolated brush border membranes of the intestinal epithelial cell were labeled with a hydrophobic photoactive compound [¹²⁵I]iodonaphthylazide. High incorporation of the radioactive naphthylazide was noted for molecular weight bands of 99 000, 86 000, 65 000, 54 000 and 30 000. Minimal labeling occurred in the higher bands of 300 000, 135 000, 125 000 and 17 000. The iodonaphthylazide label was not removed by extensive papain digestion whereas chloramine T iodinated membranes released radioactivity under the same conditions. Neither enzymatic nor transport activities were inhibited by the presence of iodonaphthylazide or the irradiation process. On the basis of the presented data it is concluded that the iodonaphthylazide unspecifically labels those portions of membrane proteins which are inserted into the lipid bilayer matrix.     

Fusion and lipid exchange in vesicles containing lipophilic spin labels

Lipophilic non-electrolyte spin labels greatly accelerate the fusion of unilamellar vesicles of dipalmitoylphosphatidylcholine when the system is maintained below the lipid phase transition. Differential scanning calorimetry and centrifugation measurements show that the transformed vesicles are large and probably unilamellar. Differential scanning calorimetry and fluorescence depolarization measurements were also carried out on mixtures of labeled dipalmitoylphosphatidylcholine vesicles and of vesicles composed of pure dimyristoylphosphatidylcholine. A mixing of the membrane components is observed when the vesicles are incubated above the transition temperature of the two constituent lipids. However, the process does not involve a real fusion of the entire vesicles. An exchange of lipid and label monomers between the two lipid phases seems to occur. These observations are discussed in view of the molecular organization of the spin label within the dipalmitoylphosphatidylcholine matrix below and above the lipid transition temperature.     

Subcellular localization of components of the dystrophin glycoprotein complex in cultured retinal muller glial cells

The dystrophin glycoprotein complex (DGC) is a membrane-associated protein complex binding extracellular matrix (ECM) molecules, such as laminin and forming a bridge towards the cytoskeleton. The molecular composition of the DGC is cell type dependent and it is involved in cell adhesion and motility. Here we present immunocytochemical localization of beta-dystroglycan, the central member of the DGC, utrophin and Dp71f, the spliced 71 kDa dystrophin protein product of the DMD gene, in cultured retinal Muller glial cells. It is shown that beta-dystroglycan and utrophin are colocalized in clusters in all parts of Muller cells including the lamellipodium and leading edge of migrating cells. As a contrast, Dp71f labels are distinct from beta-dystroglycan and confined to the perinuclear cytoplasm of Muller cells indicating that Dp71f is not a member of the DGC in cultured Muller cells.     

RNA labeling, conjugation and ligation

Advances in RNA nanotechnology will depend on the ability to manipulate, probe the structure and engineer the function of RNA with high precision. This article reviews current abilities to incorporate site-specific labels or to conjugate other useful molecules to RNA either directly or indirectly through post-synthetic labeling methodologies that have enabled a broader understanding of RNA structure and function. Readily applicable modifications to RNA can range from isotopic labels and fluorescent or other molecular probes to protein, lipid, glycoside or nucleic acid conjugates that can be introduced using combinations of synthetic chemistry, enzymatic incorporation and various conjugation chemistries. These labels, conjugations and ligations to RNA are quintessential for further investigation and applications of RNA as they enable the visualization, structural elucidation, localization, and biodistribution of modified RNA.     

Kinase Identification with Supervised Laplacian Regularized Least Squares

Phosphorylation is catalyzed by protein kinases and is irreplaceable in regulating biological processes. Identification of phosphorylation sites with their corresponding kinases contributes to the understanding of molecular mechanisms. Mass spectrometry analysis of phosphor-proteomes generates a large number of phosphorylated sites. However, experimental methods are costly and time-consuming, and most phosphorylation sites determined by experimental methods lack kinase information. Therefore, computational methods are urgently needed to address the kinase identification problem. To this end, we propose a new kernel-based machine learning method called Supervised Laplacian Regularized Least Squares (SLapRLS), which adopts a new method to construct kernels based on the similarity matrix and minimizes both structure risk and overall inconsistency between labels and similarities. The results predicted using both Phospho.ELM and an additional independent test dataset indicate that SLapRLS can more effectively identify kinases compared to other existing algorithms.     

Phosphorescence quenching as an approach for estimating localization of triplet label in cotton fibers

The method based on the qualitative investigation of chromophore fluorescence (phosphorescence) quenching for instance, by stable nitroxide radical was first used to measure the depth of immersion of triplet label in cotton fiber as a molecular object. The concept of dynamic quenching of fluorescence in solutions and the empirical dependence of the parameters of static quenching between centers with fixed distances were used. The erythrosine triplet labels were incorporated in cotton fibers with subsequent measurement of the efficiency of label phosphorescence quenching and determination of temperature dependence of phosphorescence duration. Using above mentioned approach it became possible for the first time to estimate the depth of immersion of chromophore fragment of the labels (7.4-7.8 A) and study their molecular dynamics in the millisecond range of correlation times. Subtle differences in microstructure and molecular dynamics of the investigated samples were revealed. The proposed approach can be used for investigation of widespread biological and nonbiological objects.     

Cartilage fucoproteins with sites for cross-linking by transglutaminase

Slices of various types of cartilage were incubated with either L-[6-3H]fucose or [1,4-3H(N)]putrescine. Homogenization of the slices and fractionation of the homogenates showed for both labels that an insoluble collagenase-resistant fraction had the highest specific activity (dpm/mg dry weight). Examination of an exhaustive proteolytic digest of this insoluble fraction by ion-exchange high performance liquid chromatography showed the presence of gamma-glutamyl[3H]putrescine. Chromatography of solubilized [3H]fucoprotein fractions showed the presence of several low molecular weight peaks, as well as high molecular weight material. Incubation of [3H]fucoprotein extracts with transglutaminase increased the high molecular weight peaks and decreased the low molecular weight ones. Incubation of the cartilage slices with L-[3H]fucose plus 0.5 mM dansylcadaverine, an inhibitor of transglutaminase, caused a decrease in the insoluble and high molecular weight fraction relative to the low molecular weight peaks. It is hypothesized that this is due to inhibition of cross-link formation between fucoprotein components of the cartilage which are transglutaminase substrates. One major low molecular weight peak, which labels with both fucose and putrescine, corresponds in size with the 15,000 subunit of collagen III aminopropeptide, which is known to be a substrate for transglutaminase.     

Structure and intramolecular dynamics of a photoactive pigment-protein eosin-casein complex

The structure of eosin--casein complex was studied by triplet label method. Quantitative data on the quantum--mechanic exchange interaction between eosin centres and external quenchers were obtained. The dynamic state of water--protein matrix at -20 degrees to -180 degrees C with eosin as fluorescence and phosphorescence labels and natural chromophores of protein--tryptophane was studied.     

Interaction of a phenolic inhibitor with photosystem II particles

Photosystem II particles have been prepared from spinach and Chlamydomonas reinhardii CW 15 thylakoids. Photosynthetic electron transport in these particles is inhibited by phenolic compounds like dinoseb, but not by atrazine and diuron. The labeling patterns obtained by photoaffinity labels derived from either atrazine (azido-atrazine) or the phenolic herbicide dinoseb (azido-dinoseb) were compared in photosystem II particles and thylakoids. Whereas azido-atrazine in thylakoids of spinach as well as of Chlamydomonas labels a 32-kilodalton peptide, this label does not react in photosystem II particle preparations. Azido-dinoseb, however, labels both the thylakoid membranes and the particles, predominantly polypeptides in the 40-53 kilodalton molecular weight region. Since the latter polypeptides are probably part of the reaction center of photosystem II, it is suggested that phenolic compounds have their inhibition site within the reaction center complex. This indicates that the atrazine-binding 32-kilodalton peptide is either absent or functionally inactive in photosystem II particles, whereas the phenol inhibitor-binding peptides are not.     

PELDOR analysis of enzyme-induced structural changes in damaged DNA duplexes

PELDOR (pulsed electron-electron double resonance) spectroscopy was applied to determine spin-spin distances in spin-labeled DNA duplexes (13-mer and 17-mer) containing the damaged sites 8-oxoguanine or uncleavable abasic site analogue tetrahydrofuran. The lesions were located in one strand of the DNA, and two nitroxyl spin labels were attached at the 5'- and 3'-ends of the complementary strand. PELDOR data allow us to obtain distances between the two spin labels in DNAs, which turned out to be around 5 nm for the 13-mer DNA and around 6 nm for 17-mer DNA. Results of PELDOR measurements were supported by molecular dynamics calculations. Study of the interaction of DNA fragments with DNA repair enzyme 8-oxoguanine-DNA glycosylase from E. coli (Fpg protein) showed that this interaction leads to a noticeable decrease of the distance between spin labels, which indicates the enzyme-induced bending of the DNA duplex. This bending may be important for the mechanisms of recognition of damaged sites by DNA repair enzymes.     

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, environmental/agricultural/biodefense monitoring, nanobiotechnology, and more. For diagnostic applications, monitoring (detecting) the presence, absence, or abnormal expression of targeted proteomic or genomic biomarkers found in patient samples can be used to determine treatment approaches or therapy efficacy. In the research arena, information on molecular affinities and specificities are useful for fully characterizing the systems under investigation.Many of the current systems employed to determine molecular concentrations or affinities rely on the use of labels. Examples of these systems include immunoassays such as the enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) techniques, gel electrophoresis assays, and mass spectrometry (MS). Generally, these labels are fluorescent, radiological, or colorimetric in nature and are directly or indirectly attached to the molecular target of interest. Though the use of labels is widely accepted and has some benefits, there are drawbacks which are stimulating the development of new label-free methods for measuring these interactions. These drawbacks include practical facets such as increased assay cost, reagent lifespan and usability, storage and safety concerns, wasted time and effort in labelling, and variability among the different reagents due to the labelling processes or labels themselves. On a scientific research basis, the use of these labels can also introduce difficulties such as concerns with effects on protein functionality/structure due to the presence of the attached labels and the inability to directly measure the interactions in real time.Presented here is the use of a new label-free optical biosensor that is amenable to microarray studies, termed the Interferometric Reflectance Imaging Sensor (IRIS), for detecting proteins, DNA, antigenic material, whole pathogens (virions) and other biological material. The IRIS system has been demonstrated to have high sensitivity, precision, and reproducibility for different biomolecular interactions [1-3]. Benefits include multiplex imaging capacity, real time and endpoint measurement capabilities, and other high-throughput attributes such as reduced reagent consumption and a reduction in assay times. Additionally, the IRIS platform is simple to use, requires inexpensive equipment, and utilizes silicon-based solid phase assay components making it compatible with many contemporary surface chemistry approaches.Here, we present the use of the IRIS system from preparation of probe arrays to incubation and measurement of target binding to analysis of the results in an endpoint format. The model system will be the capture of target antibodies which are specific for human serum albumin (HSA) on HSA-spotted substrates.     

Label‐free spectroscopic tissue characterization using fluorescence excitation‐scanning spectral imaging

Spectral imaging approaches provide new possibilities for measuring and discriminating fluorescent molecules in living cells and tissues. These approaches often employ tunable filters and robust image processing algorithms to identify many fluorescent labels in a single image set. Here, we present results from a novel spectral imaging technology that scans the fluorescence excitation spectrum, demonstrating that excitation‐scanning hyperspectral image data can discriminate among tissue types and estimate the molecular composition of tissues. This approach allows fast, accurate quantification of many fluorescent species from multivariate image data without the need of exogenous labels or dyes. We evaluated the ability of the excitation‐scanning approach to identify endogenous fluorescence signatures in multiple unlabeled tissue types. Signatures were screened using multi‐pass principal component analysis. Endmember extraction techniques revealed conserved autofluorescent signatures across multiple tissue types. We further examined the ability to detect known molecular signatures by constructing spectral libraries of common endogenous fluorophores and applying multiple spectral analysis techniques on test images from lung, liver and kidney. Spectral deconvolution revealed structure‐specific morphologic contrast generated from pure molecule signatures. These results demonstrate that excitation‐scanning spectral imaging, coupled with spectral imaging processing techniques, provides an approach for discriminating among tissue types and assessing the molecular composition of tissues. Additionally, excitation scanning offers the ability to rapidly screen molecular markers across a range of tissues without using fluorescent labels. This approach lays the groundwork for translation of excitation‐scanning technologies to clinical imaging platforms.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for identifying the composition of labeled proteins

Labeled proteins are extensively used in molecular biology and environmental science. The determination of the composition and label ratio is very important for monitoring the efficiency of their separation and purification. In this paper a novel method based on matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry was developed for this purpose. The results obtained for three commercial labeled proteins showed that they are mixtures of different conjugates. In some cases, the label ratio obtained by UV spectrometry and MALDI mass spectrometry was strikingly different. For fluorescent labels such as fluorescein isothiocyanate, MALDI mass spectrometry determines the number of covalently bound labels, whereas UV absorption yields both bound and adsorbed labels. For biotinylated proteins, label ratios obtained by the 4-hydroxyazabenzene-2'-carboxylic acid (HABA)-avidin method were found to be much smaller those determined by MALDI mass spectrometry. The HABA-avidin method may therefore not be suitable for the determination of biotin label ratios.     

Optimization of immunolabeled plasmonic nanoparticles for cell surface receptor analysis

Noble metal nanoparticles hold great potential as optical contrast agents due to a unique feature, known as the plasmon resonance, which produces enhanced scattering and absorption at specific frequencies. The plasmon resonance also provides a spectral tunability that is not often found in organic fluorophores or other labeling methods. The ability to functionalize these nanoparticles with antibodies has led to their development as contrast agents for molecular optical imaging. In this review article, we present methods for optimizing the spectral agility of these labels. We discuss synthesis of gold nanorods, a plasmonic nanoparticle in which the plasmonic resonance can be tuned during synthesis to provide imaging within the spectral window commonly utilized in biomedical applications. We describe recent advances in our group to functionalize gold and silver nanoparticles using distinct antibodies, including EGFR, HER-2 and IGF-1, selected for their relevance to tumor imaging. Finally, we present characterization of these nanoparticle labels to verify their spectral properties and molecular specificity.     

表面等离子体共振技术在分子生物学中的应用

表面等离子体共振(SPR)技术可以实时、原位地测定生物分子间的相互作用而无需任何标记,可以连续监测吸附和解离过程,并可以进行多组分复合物的相互作用的研究。SPR技术在DNA的复制和转录、DNA的修复、核酸与药物的作用以及肽库和抗体库的筛选等分子生物学领域的应用研究取得了令人瞩目的进展,显示了常规技术无法比拟的优越性。