I01 The Perspective of Mass Spectrometry in Proteomics and protein's post-translational modifications

Chung-Lin Liao

Genomics Research Center, Academia Sinica
128 Academia Rd. Sec. 2 Nankang Dist.
Taipei, Taiwan

 

Recent developments in technology and instrumentation have made mass spectrometry the method of choice for identification of proteins. Current mass spectrometry platform adopting shotgun proteomics approach with collisional activation dissociation (CAD) to facilitate sequence information becomes the major tool for analyzing gel-based spotted samples and total cell lysate. The CAD process severely restricts the detection and location of protein¡¦s post-translational modifications (PTM), such as phosphorylation and glycosylation. New mass spectrometry-based methods involving electron capture dissociation (ECD), electron transfer dissociation (ETD) mechanism result in extensive peptide fragmentation that is indifferent to either peptide sequence or the presence of labile PTMs. A combination of bottom up approach (peptide level) with top down approach (intact protein) to elucidate protein¡¦s PTM will be demonstrated.

 

 

I02

Functional proteomic studies of shrimp white spot syndrome virus structural
proteins and their interactors

Chu-Fang Lo

Institute of Zoology, National Taiwan University

 

White spot syndrome virus (WSSV), is a large enveloped, double-stranded DNA virus that attacks cultured shrimp and many other crustaceans. Although many WSSV ORFs do not resemble any known proteins, the entire WSSV genome has been sequenced and shrimp EST database has been established, so a proteomic approach can now be used.

Using proteomic methods, at least forty viral structural proteins have now been identified. These structural proteins protect the viral genetic material and are also important in infecting and then transmitting the genetic material into the host cells as well as other roles in the life cycle of the virus. After proteomics identified these proteins, it was then used to determine the protein roles and/or their interactors. Several strategies have been used successfully combined with a proteomic approach, including Virus Overlay Protein Binding Assay (VOPBA), yeast two-hybrid, antibody-based methods, and affinity chromatography. Thus biochemical fractionation, immunoelectron microscopy and immuno-blotting were used to show that WSSV virion in fact has an intermediate structural layer (the tegument) instead of only outer and inner components (envelope and nucleocapsid ) as was previously thought. In combination with proteomics, yeast two-hybrid to show that VP53A interacts with chitin binding protein. Meanwhile, VOPBA has shown that VP28 has three candidate interactors, at least one of which was confirmed by GST pull-down, and a neutralization test has shown that this interaction is important for WSSV infection. We have also confirmed that the structural protein VP38B has anti-apoptotic activity.

Proteomic methods have also been used in combination with VOPBA to study non-structural proteins in 2-D gels. Candidates interacts of these proteins include 2-macroglobulin, hemocyte kazal-type proteinase inhibitor, H2B histone and cyclic AMP-regulated protein like protein.

 

 

I03 Mechanism of fibril formation by the gastric cancer-related GISP-like proteins

Yuan-Chao Loua, Meng-Ru Hoa,b, Wen-Chang Lina, Chinpan Chena

aInstitute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan, ROC; bInstitute of Bioinformatic and Structural Biology, National Tsing Hua University, Hsinchu 300, Taiwan, ROC.

 

Gastric cancer (GC) is the most prevalent malignant neoplasm and the leading cause of cancer death in many countries. However, the prognosis of GC patients is dismal, emphasizing the necessity for prevention, early detection, and better treatment for GC patients. A novel gene of GISP (GastroIntertinal Secretory Protein) was identified and suggested to have important clinical significance and applications for gastric cancer by our collaborator. Three genes related to GISP that include LITs (lithostathines), PAPs (pancreatitis-associated proteins), and PBCGF (pancreatic beta cell growth factor) were further found based on sequence comparison. These GISP-like proteins all share a common C-type animal lectin motif. Both human LIT and PAP proteins were identified in pathognomonic lesions of Alzheimer¡¦s disease (AD), indicating that they may be involved in AD. Furthermore, both human LIT and PAP were found to form fibrillar aggregates at neutral pH. By contrast, fibrils could not be seen on GISP. To gain insight into the mechanism of fibril formation by GISP-like proteins, we have applied a variety of biophysical experiments for structural studies. In this presentation, NMR solution structure of human PAP and its conformational changes associated with globular to amyloid fibril transformation will be discussed and so do the preliminary studies of other GISP-like proteins. These biophysical studies on GISP-like proteins may provide valuable information for designing and searching the lead compounds for gastric cancer therapeutics as well as for the diseases related to the amyloid fibrils.

 

 

I04 The studies of the low-resolution structural and functional studies for the fidelity of DNA polymerases in solution

Joseph Tang

The Genomic Research Center, Academia Sinica, Taiwan, and Department of Chemistry, the Ohio State University, U.S.A

 

DNA gets damaged by various environmental influences (heat, radiation, oxidative stresses and others). Fortunately, there are repair mechanisms that can correct these defects. The accuracy (fidelity) of inserting a correct nucleotide (dNTP) into the gap on the damaged site by DNA polymerases is essential for maintaining the genomic stability and preventing cancer-related mutagenesis. To investigate the molecular mechanism that leads to the nucleotide selection (substrate specificity) of DNA polymerases, we have combined the small-angle scattering (SAS) and stopped-flow fluorescence analyses for the structure-function studies of the mammalian DNA polymerase £] (Pol £]). Pol £] is a multi-subdomain protein whose subdomains contributed to different roles in the base excision repair (BER) pathway in the event of DNA damages, and recent medical studies have shown that more than 30% of tumors express variants of Pol £]. Among these mutants, I260Q has tighter DNA binding affinity than the wild type and shown very poor selection on incorporation of nucleotide (behaves as a mutator).

In our scattering studies of wild-type of Pol £], no changes of the scattering curves was detected upon incorporations of the incorrect dNTP onto the (E¡EDNA) binary complex, and only the correct dNTP insertions can induce the closed form of the (E¡EDNA¡EdNTP) ternary complex. In contrast, similar and more compact conformation of the ternary complexes was achieved by I260Q with the correct and incorrect nascent base pair. Our experimental scattering results are in good-to-excellent agreements with the known crystal structures of Pol £]. Moreover, our parallel stopped-flow fluorescence studies have also suggested that I260Q incorporates incorrect dNTP much more efficient than wild type of Pol £]. Taken together, our studies imply that the formation of the closed (active) conformation of the ternary complex of Pol £] can be introduced even without the presence of correct dNTP (favor substrate), and suggest that the substrate selectivity of Pol £] is unlikely to be enhanced exclusively by the ¡§induced-fit¡¨ mechanism.

 

 

I05 FLIM/FRET__ a novel tool for molecular dynamics imaging

Fu-Jen Kao

Institute of Biophotonics, National Yang-Ming University, Taipei, Taiwan, ROC.

 

In this post-genome era, visualizing and quantifying protein-protein interactions is a recent trend in biomedical imaging. The ability to see the dynamics behavior of a specific protein inside the living cells and tissues became possible through the application of fluorescence (Föster) resonant energy transfer (FRET) technique. Applications of the modern photonics technology allow biomedical analysis at single molecule and nanometer scale with sub-nanosecond time resolution. The integration of fluorescence spectroscopy added a new dimension to light microscopy that enables the investigation of molecular behavior. Such need, in particular, has been successfully met by the fluorescence resonance energy transfer (FRET), formulated by Förster back in 1948 and reborn again recently due to the advances of newly developed genetically encoded fluorescent labels and sensors. FRET is a non-radiative quantum-mechanical dipole-dipole interaction process whereby energy from an excited donor/fluorophore is transferred to an acceptor. FRET occurs when both donor and acceptor have sufficiently large spectral overlap, a favorable dipole-dipole orientation, proximity of 1-10nm and a large enough quantum yield. The dependence of the coupling efficiency varies with the inverse sixth power of the distance between the acceptor and donor and is designated as the Föster radius, R0 (distance at which the efficiency of energy transfer is 50%). Upon energy transfer, the fluorescence intensity and the lifetime of the donor reduce as its excited state population is being depleted. FRET measurements in a microscopic object can be conveniently carried out with fluorescence lifetime imaging microscopy (FLIM).
Fluorescence lifetime measurement is independent of changes in probe concentration, excitation intensity and other factors that limit intensity-based steady-state measurements. To record fluorescence lifetime gated image intensifiers, modulation techniques and gated photon counting can be used. In particular, state-of-the-art TCSPC systems reach count rates in the MHz range and therefore are able to record decay functions within a few milliseconds. TCSPC method has high detection efficiency, a time resolution limited only by the transit time spread of the detector, and directly delivers the decay functions in the time domain. In combination with multiphoton excitation, one can probe a limited voxel volume as small as a few femtoliters that contains single molecules.
A broad range of biological problems, including proteins interaction, conformation and functions, can thus be investigated with the multiphoton FLIM/FRET microscopy. Here we present the overview and preliminary results of the Dengue virus core protein (DEN2C) dimerisation study in HeLa cells by means of the multiphoton laser scanning-based FLIM/FRET imaging system, established at the National Yang-Ming University. Additionally, we believe FLIM/FRET is a powerful tool in addressing the following modes of interactions between bio-molecules:
    1. Cell-to-Cell Signaling: legend/receptor
    2. Immune System: antigen-antibody interactions
    3. Expression of Genes: DNA/regulator protein interactions
    4. Metabolism: Enzyme complex formation
    5. Cell Division: DNA hybridization

 

 

I06 Micro- and Nano-Radiology for Life Sciences

Yeu-Kuang Hwu

Institute of Physics, Academia Sinica, Taipei 115, Taiwan, ROC

 

With the rapid development of many biomedical imaging modalities, radiography remains one of the most used imaging methods. On top of its popularity, radiology has also benefited from the new technology capability and recently reached a new level of sophistication and power. With higher coherence of the X-ray source produced by synchrotron, phase difference is added to the absorption as anther, yet much improved, contrast mechanism. Together with the improvement in the detecting system and the capability of fabricate x-ray optic elements with nanometer precision, we can now looking deep inside into matters with unprecedented precision and speed. Sub-£gm resolution radiograph can now be obtained with ease on animals in vivo and fast phenomenon can be observed in a real time fashion. A lateral resolution of 30nm has been demonstrated using nanofabricated x-ray phase zone plate. Phase contrast is implemented in all aspect of the x-ray imaging with great success in revealing sub-cellular structures. The application of this new tool has already helped researches in materials science, biology and medicine. Ongoing developments in the instrumentation and reconstruction algorithms have generated even higher excitement with the recent demonstration of nanometer scale resolution in 3D. It is anticipated that with the new installation of X-ray free electron laser, X-rays can be used to ¡§image¡¨ a single protein molecule with atomic resolution in the near future and to continue the legacy started by Rontgen. Review of this advent of this technology, its application to various domains in science, particularly the relevance to the medicine, nano-science and -technology, and the future potential will be emphasized.

 

 

I07 Medical Imaging Experiments at SPring-8

Naoto YAGI

SPring-8/JASRI, Hyogo, Japan

 

SPring-8 is the largest synchrotron radiation facility in the world, with a circumference of 1.4 km. It is also the one with the highest energy (8 GeV). Thus, it is suitable for high resolution x-ray imaging experiments on biological samples. SPring-8 has an excellent facility for animal experiments, which makes it possible to work on live animals.

The most popular imaging techniques are angiography, tomography and phase-contrast imaging. Spatial resolution of 0.5 - 20 microns is usually used. Various types of biological samples, heart, lung, brain, kidney, liver, bone, etc. from various animals and human have been studied. Angiography and phase-contrast imaging experiments are often made in real time. For imaging at high resolution, an undulator beamline such as BL20XU or BL47XU is used because it provides a well-collimated monochromatic beam with high flux (up to 10^13 cps in a beam 0.2x1 mm^2). On the other hand, when a larger field of view is required, a bending magnet beamline (BL20B2) is used, which provides a beam as large as 25x300 mm^2. At both beamlines, the energy range is 8 - 100 keV. In order to cover the wide range of spatial resolution, several different types of x-ray detectors (x-ray television, phosphor-CCD detector, CMOS flatpanel detector) have been developed.

As examples, 3D tomography of mouse lung (by Sera et al.) and a real time refraction-contrast imaging of air penetration in lung of a newborn rabbit (by Hooper et al.) will be presented.

 

 

I08 Systems analysis of the extremely halophilic archaeon Halobacterium sp. NRC-1:
Getting biological stories from proteomics data

Rueichi R. Gana, Yu-chieh P. Kaob, Eugene C. Yic,d, Yulun Chiua, Hookeun Leee, Timothy H.Wub, Ruedi Aebersoldc,e, David R. Goodlettf, and Wailap Victor Ngb,g,h

a Institute of Biochemistry, b Institute of Bioinformatics, g Institute of Biotechnology in Medicine,
and hDepartment of Biotechnology and Laboratory Science in Medicine, National Yang Ming
University, Taipei, Taiwan, Republic of China
c Institute for Systems Biology, Seattle, Washington 98103, USA
d Zymogenetics, Inc., Seattle, Washington 98012, USA
e Institute for Molecular Systems Biology, ETH Honggerberg and Faculty of Natural Sciences,
University of Zurich, Zurich CH-8093, Switzerland
fDepartment of Medicinal Chemistry, University of Washington, Seattle, Washington 98195, USA

 

Mass spectrometry is a valuable technology for revealing the macromolecules at the terminus of the bioinformation. After the completion of the Halobacterium sp. NRC-1 genome sequence, we have initiated several proteome analyses using high-throughput liquid chromatography coupled to electrospray ionization tandem mass spectrometry so as to obtain the essential expression information for making approximate predictions of the biochemical reactions occurred in standard laboratory growth conditions. With the increased number of protein identifications in our recent studies, it becomes necessary to analysis the complex data with more efficient novel techniques. Thus we developed the BMSorter and MSNetwork tools to carry out biomodular analysis which use network information to analyze proteomics data and predict interactions between systems. As an example, an integrated analysis of the enzymes in the amino acid metabolism and citrate cycle systems found up to eight amino acids may be converted to oxaloacetate, fumarate, or oxoglutarate in the citrate cycle for energy production. The analysis also suggested the interconversions of acidic amino acids to meet the high demand of the building blocks for the acidic proteome. The details of such analysis will be discussed in details in the presentation.

 

 

I09 How winged helix/forkhead proteins using conserved residues to recognize diverse DNA sequence

Chwan-Deng Hsiao1, Kuang-Lei Tsai1,2, Cheng-Yang Huang1, Chia-Hao Chang3, Yuh-Ju Sun2, and Woei-Jer Chuang3

1Institute of Molecular Biology, Academia Sinica, Taipei, 115, Taiwan, 2Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, 300, Taiwan, and 3Department of Biochemistry, National Cheng Kung University, College of Medicine, Tainan 701, Taiwan

 

Winged helix/forkhead proteins have shown to have similar binding specificity to the core sequence. In addition, these proteins have conserved amino acid sequences in the putative recognition helix. This raises an intriguing question as how proteins use conserved residues to recognize distinct core sequences. To date, more than 200 winged helix/forkhead proteins have been identified but only two forkhead protein/DNA complexes have been reported. Prior to this study, little is known about how winged helix/forkhead proteins recognize diverse DNA sequence. We report here a 3-D crystal structure of human FOXK1a bound to a 16-base pair DNA duplex containing promoter sequence. This complex structure provides a new insight into that how the DNA-binding specificities of winged helix/forkhead proteins may be regulated by their less conserved regions. The present structural study also offers the first view of a cooperative binding of ILF to DNA that contains the core sequence. The cooperativity of ILF can arise through DNA conformability in the absence of strong protein-protein interactions. The structural evidence present here was also consistent with our biochemical data.

 

 

I10 How Do Two Membrane Proteins Talk to Each Other: Signal Relay From A Photosensory Receptor, NpSRII to Its Partner Protein, NpHtrII

Chii-Shen Yang* and John L. Spudich#

*Department of Biochemical Science and Technology, National Taiwan University
# Department of Biochemistgry and Molecular Biology, University of Texas-Huston, USA.

 

The Natronobacterium pharaonis HtrII (NpHtrII), transducer interacts with its cognate photoactive sensory rhodopsin receptor, SRII, to mediate phototaxis responses. HtrII was shown to have two transmembrane (TM) helices and forms a homodimer. To investigate how the photo-activated SRII relays activation signal to HtrII, a series of single cysteines were substituted into an engineered cysteine-less HtrII at 38 positions in its two transmembrane domains. Oxidative disulfide cross-linking efficiencies of the monocysteine mutants were measured with or without photoactivation of SRII. The results reveal conformationally active regions in the TM2 of HtrII and a face along the length of TM2 that becomes more available for cross-linking upon receptor photoactivation. One TM2 mutants, G83F, showed loss of phototaxis responses, which indicates Gly83 is critical for maintaining the proper conformation of HtrII for signal relay from the photoactivated receptor. Furtmore, the cytoplasmic loops of SRII and the membrane- proximal cytoplasmic domain of its bound transducer HtrII were examined in the dark and in the light activated state by fluorescent probes and cysteine crosslinking. Light decreased the accessibility of E-F loop position 154 in the SRII-HtrII complex, but not in free SRII, consistent with HtrII proximity, which was confirmed by tryptophans placed within a 5-residue region identified in the HtrII membrane-proximal domain that exhibited fluorescence resonance energy transfer (FRET) to an fluorescent acceptor at position 154 in SRII. The energy transfer was eliminated in the signaling deficient HtrII mutant G83F without loss of affinity for SRII. Finally, the presence of SRII and HtrII reciprocally inhibit homodimer disulfide cross-linking reactions in their membrane-proximal domains, showing that each interferes with the others self-interaction in this region. A mechanism is thus proposed in which photoactivation alters the SRII-HtrII interaction in the membrane-proximal region during the signal relay process.

 

 

I11 Single molecule dynamics of Triplex DNA:
Direct visualization and measurement.

Chia-Sheng Chang1*, Chia-Ching Chang1,2,3*, Po-Yen Lin1, Yen-Fu Chen1, and Lou-Sing Kan4*

1Institute of Physics, Academia Sinica. 2Department of Biological Sciences and Technology, National Chiao Tung University. 3National Nano Device Laboratories. 4Institute of Chemistry, Academia Sinica

 

Atomic force microscopy (AFM) has been recognized as a powerful tool to both image and manipulate a single molecule in either air or solution environment. Meanwhile, fluorescence images given rise from a pair of molecular beacons are regularly used to study the dynamic behaviors of a DNA¡¦s formation/dissociation. In this study, we have combined these two techniques to directly monitor and measure the dissociation dynamics of a DNA triplex in the single molecule level. During a DNA pulling process by AFM, single-molecule fluorescence images reveal the physical distance between the Hoogsteen and Watson strands of DNA triplex while the force curve of AFM denotes the dissociation mode of the triplex DNA, which takes a two-state process.

 

 

I12 Positrom Emission Tomography for Biomolecular Imaging

Ren-Shyan Liu

National Yang-Ming University Medical School National PET/Cyclotron Center Taipei Veterans General Hospital, Taiwan

 

Biomolecular imaging is defined as the characterization and measurement of biological processes in living animals, model systems, and humans at the cellular and molecular level using imaging detectors. The goal of biomolecular imaging is to understand the dynamics, kinetics and the grade of misregulation of biological and biochemical processes in vivo, in order to obtain relevant information for the diagnosis and treatment of a disease and for predictions about the efficacy of a therapy. Disease is a biological process in which molecular errors cause failure of the normal, well-regulated function of cells. Molecular imaging provides the means to examine individual organ systems for these molecular errors of disease. Within the general contex of nuclear medicine, positron emission tomography (PET) offers advantages of high sensitivity, use of physiological elements and accurate quantification. In recent years it has been shown that PET is capable of obtaining in vivo metabolic images of small animals with excellent image resolution and high sensitivity.
There are three molecular imaging strategies, namely direct, indirect, and surrogate. Direct molecular imaging involves direct-target interaction, which is based on imaging the target directly with a target-specific probe. PET inherently use the image-enhancing radiopharmaceuticals that are synthesized at sufficiently high specific activity to enable use of tracer concentrations of the compound (picomolar to nanomolar) for detecting molecular signals. The tracer technologies strategically provide high sensitivity for imaging small-capacity molecular systems in vivo (receptors, enzymes and transporters) at a cost of lower spatial resolution than other technologies. Biocompatible nanotechnology promises to expand the horizon for molecular imaging with novel agents, such as nanoparticles (liposomes or emulsions), dendrimers, viral constructs, buckyballs, or various polymers labeled with radionuclides to enable detection with standard imaging equipment.
Prospects appear bright for molecular imaging and the development of biocompatible nanotechnologies. Complete characterization of the human genome and the subsequent emergence of proteomics, together with progress in basic molecular and cellular research, should continue to discover new and useful target for PET biomolecular imaging.

 

 

I13 Biological pathway and gene network construction on cancer systems biology

Hsueh-Fen Juan

Department of Life Science, National Taiwan University

 

Systems biology is a new field in biology that aims at system-level understanding of biological systems. Recent progress in the field of molecular biology enables us to obtain huge amounts of data. Furthermore, with the advent of high-throughput proteomics and microarray technologies, the study of systems biology has become possible. The hope of the rapid translation of 'genes to drugs' has foundered on the reality that disease biology is complex, and that drug development must be driven by insights into biological responses. Systems biology aims to describe and to understand the operation of complex biological systems and ultimately to develop predictive models of human disease.

In our study, we found a linear RGD is a tripeptide consisting of a flexible structure that makes the motif bind to its receptor with inefficient chelating affinity. Therefore, we designed a cyclic-RGD peptide (Tpa-RGDWPC, cRGD) with rigid skeleton to closely bind with its receptor. In this lecture, I would like to introduce our research about a novel drug (cyclic RGD) on breast cancer cells by using systems biology approach, and how to construct the biological pathway and gene network. These results provide a molecular explanation for the properties of cRGD in breast cancer cells and present a valuable in-depth description of their possible impact on breast cancer therapy.

 

 

I14 Network characteristics of essential genes in E. coli

Hsuan-Cheng Huang

Institute of Bioinformatics, National Yang-Ming University, Taipei, Taiwan

 

Recently a single-gene deletion library for all of the predicted genes of E. coli has been established and simultaneously 303 genes are identified as essential genes in LB medium. Elucidating the essentiality of these essential genes is a key to understand the system level organization of living cells. Here we present our analysis to address this issue from the perspectives of protein-protein interaction and metabolic networks. We have performed a comparison between essential and non-essential genes within an interaction network of E. coli and found that essential genes have significantly more links than the non-essential genes, validating earlier findings in budding yeast. Furthermore, other topological features such as clustering-coefficients, characteristic path lengths, diameter, and betweenness centrality are investigated to find their correlation with essential genes. Based on the known metabolic reactions of E. coli collected from the KEGG (Kyoto Encyclopedia of Genes and Genomes) and EcoCyc databases, a simple metabolic simulator is developed to reconstruct and navigate all the possible pathways and participated metabolites. In-silico gene deletion is performed to obtain the variation of metabolite distributions and examine the effect of missing links. Our results indicate that the average number of affected metabolites and reactions by deletion of essential genes are significantly higher than non-essential genes.

 

 

I15 Discovery and Mechanistic Study of Novel Inhibitors against Hepatitis C Virus Helicase

Ji-Wang Chern,,† Chien-Shu Chen, Chun-Tang Chiou,Grace Shiahuy Chen, and Ding-Shinn Chen¡±

School of Pharmacy, College of Medicine, National Taiwan University, Taipei, Taiwan, Department of Applied Chemistry, Providence University, Shalu, Taiwan, ¡±Hepatitis Research Center, National Taiwan University Hospital, Taipei, Taiwan, and Faculty of Life Science, National Yang-Ming University, Taipei, Taiwan

 

Patients with persistent Hepatitis C virus (HCV) infection are at risk of developing liver cirrhosis and hepatocellular carcinoma. The current recommended HCV treatments use interferon monotherapy or a combination of interferon and ribavirin, which have limitations due to unsatisfactory response rates and adverse side effects. Therefore, there is a great need to develop more effective agents for the treatment of HCV infection. The HCV NS3 is a bifunctional enzyme, of which the one-third of N-terminus possesses serine protease activity and the remaining two-thirds of C-terminal region has RNA helicase/NTPase activity. The crystal structures of HCV NS3 helicase have been revealed and show similar global Y-shaped conformation consisting of three domains. It was our intention to find potential HCV helicase inhibitors by blocking the closing of the interface between domains 1 (ATPase domain) and 2 (RNA binding domain).

Starting from the identification of an active dye, soluble blue HT, in our virtual docking screening, we subsequently utilized 2D substructure searches leading to the discovery of the low-molecular weight HCV helicase inhibitors, triphenylmethane derivatives, with approximately three-fold improved potency (IC50 about 13 £gM)ƒw compared to the initial hit. The unwinding ability of HCV helicase is dependent with ATP binding, ATP hydrolysis, and RNA binding. Three compounds inhibited ATPase activity of HCV NTPase/helicase with IC50 values about 25 £gM in a mixed type manner while one inhibitor had little effect on the ATPase. Furthermore, all four inhibitors were demonstrated to interfere the RNA binding. Mutational analysis of HCV helicase has suggested that interfering agents on RNA binding activity should be preferentially considered since minimal ATPase activity was sufficient for helicase activity but a reduction of RNA binding activity distinctly inhibited the helicase activity. Therefore, these inhibitors may serve as a novel class to offer a basis in the field of developing HCV helicase inhibitors.

 

 

I16 Development of Functionalized Nanoparticles as Affinity Probes for Target Proteins Enrichment and Identification

Chung-Cheng Lin

Department of Chemistry, National Tsing Hua University, Taiwan

 

Biomolecule-conjugated nanoparticles (NPs) have been demonstrated to have promising applications in bioanalysis. In this talk, I will focus on our recently progress in the development of functionalized nanoparticles and their applications in separation of target proteins. Due to its large surface area to volume ratio and homogeneity in aqueous solution, nanoparticle was demonstrated as a good carrier of affinity probe for target protein separation. The surface plasma resonance (SPR) was used to investigated the interactions between carbohydrate conjugated NPs and target proteins. The results showed that the ligand affinity with target protein was significantly enhanced when it was assembled on the nanoparticle. The MALDI-TOF MS analysis was applied to identify the protein binding epitope for the ligand. This approach was further extended to identify the biomarker in human serum.