Electron tunneling in proteins and water

The subject of this thesis is electronic coupling in donor-bridge-acceptor systems. In Chapter 2, ET properties of cyanide-bridged dinuclear ruthenium complexes were investigated. The strong interaction between the mixed-valent ruthenium centers leads to intense metal-to-metal charge transfer bands (MMCT). Hush analysis of the MMCT absorption bands yields the electronic-coupling strength between the metal centers (H_(AB)) and the total reorganization energy (λ). Comparison of ET kinetics to calculated rates shows that classical ET models fail to account for the observed kinetics and nuclear tunneling must be considered.

In Chapter 3, ET rates were measured in four ruthenium-modified highpotential iron-sulfur proteins (HiPIP), which were modified at position His50, His81, His42 and His18, respectively. ET kinetics for the His50 and His81 mutants are a factor of 300 different, while the donor-acceptor separation is nearly identical. PATHWAY calculations corroborate these measurements and highlight the importance of structural detail of the intervening protein matrix.

In Chapter 4, the distance dependence of ET through water bridges was measured. Photoinduced ET measurements in aqueous glasses at 77 K show that water is a poor medium for ET. Luminescence decay and quantum yield data were analyzed in the context of a quenching model that accounts for the exponential distance dependence of ET, the distance distribution of donors and acceptors embedded in the glass and the excluded volumes generated by the finite sizes of the donors and acceptors.

In Chapter 5, the pH-dependent excited state dynamics of ruthenium-modified amino acids were measured. The [Ru(bpy)_(3)] ^(2+) chromophore was linked to amino acids via an amide linkage. Protonation of the amide oxygen effectively quenches the excited state. In addition. time-resolved and steady-state luminescence data reveal that nonradiative rates are very sensitive to the protonation state and the structure of the amino acid moiety.

Quantitative, Time-Resolved Proteomic Analysis Using Bio-Orthogonal Non-Canonical Amino Acid Tagging

Bio-orthogonal non-canonical amino acid tagging (BONCAT) is an analytical method that allows the selective analysis of the subset of newly synthesized cellular proteins produced in response to a biological stimulus. In BONCAT, cells are treated with the non-canonical amino acid L-azidohomoalanine (Aha), which is utilized in protein synthesis in place of methionine by wild-type translational machinery. Nascent, Aha-labeled proteins are selectively ligated to affinity tags for enrichment and subsequently identified via mass spectrometry. The work presented in this thesis exhibits advancements in and applications of the BONCAT technology that establishes it as an effective tool for analyzing proteome dynamics with time-resolved precision.

Chapter 1 introduces the BONCAT method and serves as an outline for the thesis as a whole. I discuss motivations behind the methodological advancements in Chapter 2 and the biological applications in Chapters 2 and 3.

Chapter 2 presents methodological developments that make BONCAT a proteomic tool capable of, in addition to identifying newly synthesized proteins, accurately quantifying rates of protein synthesis. I demonstrate that this quantitative BONCAT approach can measure proteome-wide patterns of protein synthesis at time scales inaccessible to alternative techniques.

In Chapter 3, I use BONCAT to study the biological function of the small RNA regulator CyaR in Escherichia coli. I correctly identify previously known CyaR targets, and validate several new CyaR targets, expanding the functional roles of the sRNA regulator.

In Chapter 4, I use BONCAT to measure the proteomic profile of the quorum sensing bacterium Vibrio harveyi during the time-dependent transition from individual- to group-behaviors. My analysis reveals new quorum-sensing-regulated proteins with diverse functions, including transcription factors, chemotaxis proteins, transport proteins, and proteins involved in iron homeostasis.

Overall, this work describes how to use BONCAT to perform quantitative, time-resolved proteomic analysis and demonstrates that these measurements can be used to study a broad range of biological processes.

Development of Microfluidic Devices with the Use of Nanotechnology to Aid in the Analysis of Biological Systems Including Membrane Protein Separation, Single Cell Analysis, and Genetic Markers

Computation technology has dramatically changed the world around us; you can hardly find an area where cell phones have not saturated the market, yet there is a significant lack of breakthroughs in the development to integrate the computer with biological environments. This is largely the result of the incompatibility of the materials used in both environments; biological environments and experiments tend to need aqueous environments. To help aid in these development chemists, engineers, physicists and biologists have begun to develop microfluidics to help bridge this divide. Unfortunately, the microfluidic devices required large external support equipment to run the device. This thesis presents a series of several microfluidic methods that can help integrate engineering and biology by exploiting nanotechnology to help push the field of microfluidics back to its intended purpose, small integrated biological and electrical devices. I demonstrate this goal by developing different methods and devices to (1) separate membrane bound proteins with the use of microfluidics, (2) use optical technology to make fiber optic cables into protein sensors, (3) generate new fluidic devices using semiconductor material to manipulate single cells, and (4) develop a new genetic microfluidic based diagnostic assay that works with current PCR methodology to provide faster and cheaper results. All of these methods and systems can be used as components to build a self-contained biomedical device.

Technology for Single Cell Protein Analysis in Immunology and Cancer Prognostics

The first chapter of this thesis deals with automating data gathering for single cell microfluidic tests. The programs developed saved significant amounts of time with no loss in accuracy. The technology from this chapter was applied to experiments in both Chapters 4 and 5.

The second chapter describes the use of statistical learning to prognose if an anti-angiogenic drug (Bevacizumab) would successfully treat a glioblastoma multiforme tumor. This was conducted by first measuring protein levels from 92 blood samples using the DNA-encoded antibody library platform. This allowed the measure of 35 different proteins per sample, with comparable sensitivity to ELISA. Two statistical learning models were developed in order to predict whether the treatment would succeed. The first, logistic regression, predicted with 85% accuracy and an AUC of 0.901 using a five protein panel. These five proteins were statistically significant predictors and gave insight into the mechanism behind anti-angiogenic success/failure. The second model, an ensemble model of logistic regression, kNN, and random forest, predicted with a slightly higher accuracy of 87%.

The third chapter details the development of a photocleavable conjugate that multiplexed cell surface detection in microfluidic devices. The method successfully detected streptavidin on coated beads with 92% positive predictive rate. Furthermore, chambers with 0, 1, 2, and 3+ beads were statistically distinguishable. The method was then used to detect CD3 on Jurkat T cells, yielding a positive predictive rate of 49% and false positive rate of 0%.

The fourth chapter talks about the use of measuring T cell polyfunctionality in order to predict whether a patient will succeed an adoptive T cells transfer therapy. In 15 patients, we measured 10 proteins from individual T cells (~300 cells per patient). The polyfunctional strength index was calculated, which was then correlated with the patient's progress free survival (PFS) time. 52 other parameters measured in the single cell test were correlated with the PFS. No statistical correlator has been determined, however, and more data is necessary to reach a conclusion.

Finally, the fifth chapter talks about the interactions between T cells and how that affects their protein secretion. It was observed that T cells in direct contact selectively enhance their protein secretion, in some cases by over 5 fold. This occurred for Granzyme B, Perforin, CCL4, TNFa, and IFNg. IL- 10 was shown to decrease slightly upon contact. This phenomenon held true for T cells from all patients tested (n=8). Using single cell data, the theoretical protein secretion frequency was calculated for two cells and then compared to the observed rate of secretion for both two cells not in contact, and two cells in contact. In over 90% of cases, the theoretical protein secretion rate matched that of two cells not in contact.

Part I. Synchronization of the cell division cycle of HeLa cells in suspension cultures. Part II. Studies on chromosomal proteins of HeLa cells during the cell division cycle

Part I

These studies investigate the potential of single and double treatments with either 5-fluorodeoxyuridine of excess thymidine to induce cell division synchrony in suspension cultures of HeLa cells. The patterns of nucleic acid synthesis and cell proliferation have been analyzed in cultures thus synchronized. Several changes in cell population during long incubation with 5-fluorodeoxyuridine or excess thymidine are also described. These results are subjected to detailed evaluation in terms of the degree and quality of synchrony finally achieved.

Part II

Histones and non-histone proteins associated with interphase and metaphase chromosomes of HeLa cells have been qualitatively and quantitatively analyzed. Histones were fractionated by chromatography on Amberlite CG-50 and further characterized by analytical disc electrophoresis and amino acid analysis of each chromatographic fraction. It is concluded that histones of HeLa cells are comprised of only a small number of major components and that these components are homologous to those of other higher organisms. Of all the histones, arginine-rich histone III alone contains cysteine and can polymerize through formation of intermolecular disulfide bridges between histone III monomers.

A detailed comparison by chromatography and disc electrophoresis established that interphase and metaphase histones are made up of similar components. However, certain quantitative differences in proportions of different histones of interphase and metaphase cells are reported. Indirect evidence indicates that a certain proportion of metaphase histone III is polymerized through intermolecular disulfide links, whereas interphase histone III occurs mainly in the monomeric form.

Metaphase chromosomes are associated with an additional acid-soluble protein fraction which is absent from interphase chromosomes. All of these additional acid-soluble proteins of metaphase chromosomes are shown to be non-histones and it is concluded that the histone/DNA ratio is identical in interphase and metaphase chromosomes. The bulk of acid-soluble non-histone proteins of metaphase chromosomes were found to be polymerized through disulfide bridges; corresponding interphase non-histone proteins displayed no evidence of similar polymerization.

The factors responsible for the condensed configuration and metabolic inactivity of metaphase chromosomes are discussed in light of these findings.

The relationship between histone and DNA synthesis in nondividing differentiated chicken erythrocyte cells and in rapidly dividing undifferentiated HeLa cells is also investigated. Of all the histones, only arginine-rich histones are synthesized in mature erythrocytes. Histone synthesis in HeLa cells was studied in both unsynchronized and synchronized cultures. In HeLa cells, only part of the synthesis of all histone fractions is dependent on concurrent DNA synthesis, whereas all histones are synthesized in varying degrees even in the absence of DNA synthesis.

Estudo estrutural e funcional das proteínas ligadoras de ácidos graxos (FABP- Fatty Acid Binding Proteins) de Fasciola hepatica

As proteínas ligadoras de ácidos graxos (Fatty Acid Binding Proteins, FABPs) de parasitos têm um papel importante no processo de infecção por estes organismos. Por este motivo, estas proteínas são antígenos candidatos para vacina contra a infecção por Schistosoma mansoni e Fasciola hepatica. No presente trabalho foram caracterizadas FABPs de F. hepatica e comparadas com a proteína Sm14 de S. mansoni, a FABP de parasito melhor caracterizada, mediante análise de sequências e estruturas modeladas. Também foram clonadas, expressas e purificadas as FABPs tipo 1 e tipo 3 de F. hepatica. Os resultados do presente estudo indicam que a FABP tipo 3 de F. hepatica é relacionada estrutural, imunológica e funcionalmente com a Sm14, um candidato vacinal amplamente estudado. Devido à importância da Sm14 como alvo para o desenvolvimento de vacina para a esquistossomose, as características apresentadas pela FhFABP3 de F. hepatica apontam esta proteína como um candidato importante também para o desenvolvimento de uma vacina contra a fasciolose

Proteomic Analysis of the Ubiquitin Landscape in the Drosophila Embryonic Nervous System and the Adult Photoreceptor Cells

Background Ubiquitination is known to regulate physiological neuronal functions as well as to be involved in a number of neuronal diseases. Several ubiquitin proteomic approaches have been developed during the last decade but, as they have been mostly applied to non-neuronal cell culture, very little is yet known about neuronal ubiquitination pathways in vivo. Methodology/Principal Findings Using an in vivo biotinylation strategy we have isolated and identified the ubiquitinated proteome in neurons both for the developing embryonic brain and for the adult eye of Drosophila melanogaster. Bioinformatic comparison of both datasets indicates a significant difference on the ubiquitin substrates, which logically correlates with the processes that are most active at each of the developmental stages. Detection within the isolated material of two ubiquitin E3 ligases, Parkin and Ube3a, indicates their ubiquitinating activity on the studied tissues. Further identification of the proteins that do accumulate upon interference with the proteasomal degradative pathway provides an indication of the proteins that are targeted for clearance in neurons. Last, we report the proof-of-principle validation of two lysine residues required for nSyb ubiquitination. Conclusions/Significance These data cast light on the differential and common ubiquitination pathways between the embryonic and adult neurons, and hence will contribute to the understanding of the mechanisms by which neuronal function is regulated. The in vivo biotinylation methodology described here complements other approaches for ubiquitome study and offers unique advantages, and is poised to provide further insight into disease mechanisms related to the ubiquitin proteasome system.

Global analysis of mRNA half-lives and de novo transcription in a dinoflagellate, Karenia brevis

Dinoflagellates possess many physiological processes that appear to be under post-transcriptional control. However, the extent to which their genes are regulated post-transcriptionally remains unresolved. To gain insight into the roles of differential mRNA stability and de novo transcription in dinoflagellates, we biosynthetically labeled RNA with 4-thiouracil to isolate newly transcribed and pre-existing RNA pools in Karenia brevis. These isolated fractions were then used for analysis of global mRNA stability and de novo transcription by hybridization to a K. brevis microarray. Global K. brevis mRNA half-lives were calculated from the ratio of newly transcribed to pre-existing RNA for 7086 array features using the online software HALO (Half-life Organizer). Overall, mRNA half-lives were substantially longer than reported in other organisms studied at the global level, ranging from 42 minutes to greater than 144 h, with a median of 33 hours. Consistent with well-documented trends observed in other organisms, housekeeping processes, including energy metabolism and transport, were significantly enriched in the most highly stable messages. Shorter-lived transcripts included a higher proportion of transcriptional regulation, stress response, and other response/regulatory processes. One such family of proteins involved in post-transcriptional regulation in chloroplasts and mitochondria, the pentatricopeptide repeat (PPR) proteins, had dramatically shorter half-lives when compared to the arrayed transcriptome. As transcript abundances for PPR proteins were previously observed to rapidly increase in response to nutrient addition, we queried the newly synthesized RNA pools at 1 and 4 h following nitrate addition to N-depleted cultures. Transcriptome-wide there was little evidence of increases in the rate of de novo transcription during the first 4 h, relative to that in N-depleted cells, and no evidence for increased PPR protein transcription. These results lend support to the growing consensus of post-transcriptional control of gene expression in dinoflagellates.

Toward an understanding of the molecular mechanism for successful blood feeding by coupling proteomics analysis with pharmacological testing of horsefly salivary glands

Horseflies are economically important blood-feeding arthropods and also a nuisance for humans and vectors for filariasis. They rely heavily on the pharmacological properties of their saliva to get a blood meal and suppress immune reactions of hosts. Little information is available on antihemostatic substances in horsefly salivary glands; especially no horsefly immune suppressants have been reported. By proteomics or peptidomics and coupling transcriptome analysis with pharmacological testing, several families of proteins or peptides, which act mainly on the hemostatic system or immune system of the host, were identified and characterized from 30,000 pairs salivary glands of the horsefly Tabanus yao (Diptera, Tabanidae). They are: (i) a novel family of inhibitors of platelet aggregation including two members, which possibly inhibit platelet aggregation by a novel mechanism and act on platelet membrane, (ii) a novel family of immunosuppressant peptides including 12 members, which can inhibit interferon-gamma production and increase interleukin-10 secretion, (iii) a serine protease inhibitor with 56 amino acid residues containing anticoagulant activity, (iv) a serine protease with anticoagulant activity, (v) a protease with fibrinogenolytic activity, (vi) three families of antimicrobial peptides including six members, (vii) a hyaluronidase, (viii) a vasodilator peptide, which is an isoform of vasotab identified from Hybomitra bimaculata, and interestingly (ix) two metallothioneins, which are the first metallothioneins reported from invertebrate salivary glands. The current work will facilitate the understanding of the molecular mechanisms of the ectoparasite-host relationship and help in identifying novel vaccine targets and novel leading pharmacological compounds.

Purification and cloning of cysteine-rich proteins from Trimeresurus jerdonii and Naja atra venoms

Three 26 kDa proteins, named as TJ-CRVP, NA-CRVP1 and NA-CRVP2, were isolated from the venoms of Trimeresurus jerdonii and Naja atra, respectively. The N-terminal sequences of TJ-CRVP and NA-CRVPs were determined. These components were devoid of the enzymatic activities tested, such as phospholipase A(2), arginine esterase, proteolysis, L-amino acid oxidase, 5' nucleotidase, acetylcholinesterase. Furthermore, these three components did not have the following biological activities: coagulant and anticoagulant activities, lethal activity, myotoxicity, hemorrhagic activity, platelet aggregation and platelet aggregation-inhibiting activities. These proteins are named as cysteine-rich venom protein (CRVP) because their sequences showed high level of similarity with mammalian cysteine-rich secretory protein (CRISP) family. Recently, some CRISP-like proteins were also isolated from several different snake venoms, including Agkistrodon blomhoffi, Trimeresurus flavoviridis, Lanticauda semifascita and king cobra. We presumed that CRVP might be a common component in snake venoms. Of particular interest, phylogenetic analysis and sequence alignment showed that NA-CRVP1 and ophanin, both from elapid snakes, share higher similarity with CRVPs from Viperidae snakes. (C) 2003 Elsevier Ltd. All rights reserved.

Structure analysis of CCR5 from human and primates

It is well known that the chemokine receptor CCR5 plays very important roles in HIV-1 virus infection. A three-dimensional molecular model of human CCR5 was generated by SYBYL, a distance geometry-based homologous modeling package, using the corresponding transmembrane domain of bacteriorhodopsin as the template. On the basis of human CCR5 model, we also built 18 3D molecular models of CCR5 in primates from Pongo pygmaeus, Pygathrix nemaeus, Macaca assameniss, Trachy-pithecus phayrei, T. francoisi, M. arotoides, Rhinopithecus roxellance, R, bieti, R. avunculus, Hylobates leucogenys, Pan troglodytes, Gorilla gorilla, Cercopithecus aethiops 1, C. aethiops 2, Papio hamadryas M. mulatta, M. fascicularis and M. nemestrina. Structural analyses and statistics results suggested that the main-chains of the primate CCR5 were similar to that of the human CCR5 and that the fit-RMS deviation values of these primate CCR5 were less than 0.1 Angstrom. Moreover, the structures of these CCR5 proteins, except those of the African green monkey 1 (C.aet1), do not have a remarkable difference. It is proved that the 14th residue is possibly very important in the inhibition infections by M-tropic HIV-1, and it is also demonstrated that the 13th residue of human CCR5 was changed from asparagine into aspartic acid in all these primates. It means that the primate CCR5 no longer depend on CD4 for efficient entry, but human CCR5 may have evolved subsequently due to the use of CD4 as a receptor, allowing the high-affinity chemokine receptor-binding site of HIV to be sequestered from host immune surveillance. (C) 2000 Elsevier Science B.V. All rights reserved.

Analysis of the intein's evolution

Inteins are internal segment peptides which can self-splicing at the protein level. Although inteins are found in all three domains of life, they are not evenly distributed among species and proteins. So the evolution of intein have been capturing much at

Venomics and biological analysis of Scatophagus argus argus (Spotted scat) venom isolated in Persian Gulf, extraction and purification of hemolytic protein

Green scat namely as Scatophagus argus argus is a venomous aquarium fish belonging to Scatophagidae family. It can induce painful wounds in injured hand with partial paralysis to whom that touch the spines. Dorsal and ventral rough spines contain cells that produce venom with toxic activities. According to unpublished data collected from local hospitals in southern coastal region of Iran, S. argus is reported as a venomous fish. Envenomation induces clinical symptoms such as local pain, partial paralysis, erythema and itching. In the present study green scat (spotted scat) was collected from Persian Gulf coastal waters. SDS-PAGE indicated 12 distinct bands in the venom ranged between 10-250 KDa. The crude venom had hemolytic activity on human erythrocytes (1%) with an LC100 (Lytic Concentration) of about 1.7 μg. The crude venom can release 813 μg proteins from 0.5% casein. Phospholipase C activity was recorded at 3.125 μg of total venom. Our findings showed that the edematic activity remained over 48 h after injection. The purification of the venom was done by HPLC and 30 peaks were obtained within 80 min but only one peak in 68 min retention time showed hemolytic activity at 90% acetonitril was isolated. The area percentage of the hemolytic protein showed that this hemolytic protein consist of 32 percent of total proteins and its molecular weight was 72 KDa in SDS_PAGE. The results demonstrated that crude venom extracted from Iranian coastal border has different toxic and enzymatic activities.

Proteomic analysis of the sarcosine-insoluble outer membrane fraction of Flavobacterium columnare

Outer membrane proteins (OMPs) of bacteria are key molecules interacting with the host environment. Flavobacterium columnare, a pathogen-causing columnaris disease of fish worldwide, was studied in order to understand the composition of its OMPs. The sarcosine-insoluble membrane fraction of the OMPs was analysed using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in combination with reverse-phase high-performance liquid chromatography-tandem mass spectrometry (RP-HPLC MS/MS). Thirty-six proteins were identified, including proteins involved in cell wall/membrane biogenesis, specific transport of various nutrients and in essential metabolism. The present study is the first report on the OMPs of F. columnare, and may serve as the basis for understanding the pathogenesis of the bacterium.