Browsing by Author "Wang, Yuhong"
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Item A Genome-Wide Search for Tumor Suppressor MicroRNAs in Ovarian Cancer(2014-08) Hernandez Herrera, Anadulce 1982-; Gunaratne, Preethi H.; Flores, Elsa R.; Wang, Yuhong; Zhang, Xiaoliu Shaun; Widger, William R.Ovarian cancer is one of the most lethal cancers among women. The Cancer Genome Atlas (TCGA) is a collaborative effort, which seeks to characterize the complete set of molecular changes associated with cancer and provide a public resource that will allow the development of new therapies and better diagnostic tools for cancer. Much of the focus is on protein coding genes and our understanding of the contribution from non-coding RNAs is lagging behind. MicroRNAs are small non-coding RNAs that can bind and repress hundreds of gene targets to regulate gene networks. Therefore, defining and understanding the miRNA-regulated genes offer new insights that can be clinically applied for many of the disease. In order to identify new tumor suppressors for ovarian cancer and downstream targets that drive key aspects of this disease such as drug resistance and metastatic spread, 3 candidates were selected from the microRNA-mRNA bioinformatic analyses from the TCGA. A combination of molecular and functional studies confirmed that miR-29a that can regulate genes from the histone modifier and cell cycle pathways, inhibit proliferation and moderately increase cisplatin response in the p53-WT HEYA8; miR-509-3p which targets genes from the ECM/EMT networks, inhibits cell proliferation in p53-WT HEYA8 and p53-mut OVCAR8 and correlated with improved overall survival when analyzed by in situ hybridization in an independent cohort; miR-130b increases apoptosis by 3-fold in p53-mutant OVCAR8 and p53-wild-type HEYA8 and significantly induces TAp63 and BCL2L11 (BIM). Forced expression of TAp63 decreases cell viability by 60-80% and miR-130b-ABT-737 (BCL2L11-mimetics) combination increases apoptosis by 9-fold suggesting TAp63 and BIM are critical effectors of the tumor-suppressive mechanisms driven by miR-130b, and can be used to develop new therapeutic strategies that will target p53 WT and p53 mutant tumors.Item Amino Acid purification and Single-Molecule FRET study of tRNA translocation(2019) Kapadia, Taher; Lin, RanUnderstanding the basic translational mechanisms of the ribosome during protein translation is essential to developing novel chemical defenses against the increasing amount of anti-biotic resistant bacteria. To study the fundamentals of the protein building process, we can employ smFRET studies which require debris-free, pure samples of amino acids, the building blocks of all proteins. Our experiment focused on the overexpression and purification of the Glutamic Acid tRNA and its translocation in the ribosome. This was accomplished by inserting a Glutamic Acid sequence into a vector plasmid through a series of digestion and ligation reactions. The recombinant plasmid was then overexpressed in E. Coli and the tRNA was purified through chloroform extraction, gel filtration, and ion-exchange chromatography. The pure sample was used to make a ribosome complex and the translocation of Glutamic Acid from the A site in the ribosome to the P site was studied through smFRET. From the results, it was concluded that the ribosome can efficiently process the Glutamic Acid tRNA when a frameshifting motif is not present. This raises the question of what may occur if frameshifting motifs are added into an mRNA transcript and how this may affect the trans-locational process of glutamic acid. The future of this study should target the translational process in the presence of frameshifting motifs to identify targets which can halt the building of the proteins. Studies attempting to apply such mechanisms to cells in a host should, however, be mindful that both the bacterial and host ribosomes are susceptible to frameshifting which may limit or complicate discovery of target sites.Item Antiproliferative and pro-apoptotic actions of Estrogen receptor β in prostate cancer(2013-05) Dey, Prasenjit 1978-; Gustafsson, Jan-Åke; Ström, Anders M.; Schwartz, Robert J.; Webb, Paul; Wang, YuhongHigh Gleason grade prostate cancers are aggressive. Currently, the major target for treatment is the androgen receptor. Recent literature points towards a tumor suppressive role of estrogen receptor β (ERβ), which has a potential to be exploited as a target for novel therapeutics used for treatment of prostate cancer. In Chapter 2 of the thesis, we showed that ERβ-selective agonists elicited an increase in apoptosis and this was accompanied by an increase in expression of the pro-apoptotic factor PUMA. Induction of PUMA was dependent on the presence of the transcription factor FOXO3 but was independent of p53. In the ventral prostates of ERβ-/- mice, expression of FOXO3a is lower than that in WT littermates demonstrating a relationship between ERβ and FOXO3a expression found in PC3 and LNCaP cells. Furthermore, in prostate cancers of Gleason grade 4 or higher there was a marked reduction of both ERβ and FOXO3a, while both genes were well expressed in BPH sections. In Chapter 3 of the thesis, we investigated whether the loss of ERβ (also called as ERβ1) and/or expression of its splice variant ERβ2 affected signaling pathways involved in proliferation and bone metastasis of prostate cancer. We found repressed expression of the bone metastasis regulator Runx2 and its target gene, Slug by ERβ1. In addition, the expression of Twist1, a factor whose expression strongly correlates with high Gleason grade prostate cancer, was increased by ERβ2. In terms of cell cycle modification, of the two receptors, ERβ1, but not ERβ2, inhibited proliferation and expression of the proliferation markers Cyclin E, c-Myc, and p45Skp2. Xenograft studies using athymic nude mice confirmed the proliferative effect of ERβ2, as tumors in mice bearing PC3-ERβ2 cells were substantially larger than tumors in mice bearing PC3-control and PC3-ERβ1 cells.Item BIOFUNCTIONALIZE SURFACES AND COLLOIDS FOR REDUCING BACTERIAL ADHESION AND INFECTION(2016-12-08) Zhu, Zhiling 1986-; Cai, Chengzhi; Thummel, Randolph P.; Daugulis, Olafs; Lubchenko, Vassiliy; Wang, YuhongPrevention of pathogenic colonization on medical devices over a long period of time remains a great challenge, especially in high-nutrient environment that accelerates the production of biomass leading to biofouling of the devices. Since biofouling and the subsequent pathogen colonization is inevitable, an alternative strategy using non-pathogenic bacteria as living guards against pathogenic colonization on medical devices attracts increasing interest. Crucial to the success of this strategy is to pre-establish a high coverage and stable biofilm of benign bacteria on the surface. Silicone elastomers are one of the most widely used materials in biomedical devices. This dissertation presents the studies aiming to biofunctionalize the silicone surfaces to enhance the formation of non-pathogenic biofilms that reduce the colonization by pathogenic bacteria. The non-pathogenic Escherichia coli 83972 is sluggish to establish bladder colonization. Pre-establishing high coverage and stable E. coli 83972 biofilms on silicone catheter surfaces is promising to improve the bacterial duration in bladder as well as simplify the tedious inoculation protocol. We use a high affinity propynylphenyl mannoside (PPh-Man) ligand for binding to the non-pathogenic E. coli, which is pre-conjugated to the amino-terminated poly(amido amine) (PAMAM) dendrimers that are covalently attached to activated silicone substrates. The coverage, stability and bacterial interference efficacy of non-pathogenic biofilms on the modified silicone substrates were evaluated. As a result, 94 % non-pathogenic bacteria were retained on the modified silicone substrates under >0.5 Pa shear stress. Synthesis of a series of mannoside derivatives varying the aromatic glycosidic linkage, including CF3-BP-Man, C2F5-BP-Man, CF3-TP-Man, CH3-BP-Man, CF3-PPh-Man and CH3-PPh-Man with an OEG linker terminated with an azido group for surface immobilization via the copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC). These linkers were selected based on the reported X-ray structure-based design, and optimization of biaryl mannoside FimH inhibitors. To quantify the equilibrium binding affinity (IC50) of FimH to these mannosides, a method based on flow cytometry (FCM) was established. Mono-disperse polystyrene microspheres (~10 µm diameter) were covalently modified with mannoside, and their density was determined. These mannoside-coated particles were used for bacterial-particle incubation assay. This method greatly improved the efficiency and reproducibility over the reported method based using eukaryotic cell as target for measurement and comparison of the binding affinity of mannoside derivatives. An efficient method by using CO2 plasma to activate the catheter and functionalize the catheter inner lumen with G5 PAMAM dendrimers was developed. The efficacy of coatings was evaluated by radioactive isotopic iodine-125 labeling analysis. As a result, the PAMAM coverage on catheter is (94 ± 8) % and the PAMAM density per cm cather was (2.82 ± 0.24) × 1012 PAMAM/cm. The highest 125I-PAMAM coating was found in the innermost chamber. The best condition for CO2 plasma is 45 seconds in a low-power mode. The long-term stability result indicated more than 90 % of PAMAM dendrimers retained on the catheter rather than in the artificial urine solution for up to 15 days at room temperature statically. Our bioconjugation tool for surface and colloid functionalization is mostly based on the CuAAC reaction. The current Cu(I)-tris(triazolylmethyl)-amine catalysts still have the limitation of relatively low efficiency, oxidative degradation, and ligand dissociation in biological conditions. Contributing to address these issues, we measured kobs, KD and V0 for twenty one Cu(I) catalysts. Tris(triazolylmethyl)amines remained to be the best co-catalyst for CuAAC, but they were weaker ligands of Cu(I) compared to common biological ligands. More importantly, the polynuclear copper(I)-ligand-alkyne complexes were found crucial for CuAAC reaction that was directly observed by electrospray-ionization mass spectrometry (ESI-MS). Significant air oxidation still occurred in the presence of ligands, leading to rapid degradation of a histidine-containing peptide as a model of proteins. We present here a simple approach to reduce the air oxidation of the peptide, based on the use of oligo(ethylene glycol) chains tethered to the Cu(I) ligand for sacrificial protection of the ligand and the biomolecules from being oxidized by the oxy radicals generated from the copper center during CuAAC reaction. I also initiated the project on the development of fluorous silica nanospheres as carriers of perfluoroalkanes as ultrasound imaging agents and drug molecules with a fluorous tag for controlled release. The use of fluorous nanospheres should allow the loading of low-boiling point perfluoropropane (PFP) and perfluorobutane (PFB) that can be released and imaged by applying low frequency ultrasound (LFUS), and the fluorous-tagged cargo molecules be ejected with a temporal and spatial control.Item Developing a Magnetic-Based Force Spectroscopy with High Molecular Resolution(2014-08) De Silva, Lashan Madusha 1982-; Xu, Shoujun; Baldelli, Steven; Jacobson, Allan J.; Yang, Ding-Shyue; Wang, YuhongMagnetic materials are widely used in chemistry, biology, and medicine. Consequently, many magnetic-based techniques have been developed to facilitate these applications by providing quantity, spatial information, and molecular specificity for the targeted molecules. However, each of the three aspects represents a challenge for the techniques, namely, sensitivity, resolution, and specificity, respectively. Recently, our group has reported several new techniques for quantitative and molecule-specific detection of magnetic signals. Using an atomic magnetometer coupled with a scanning detection method, as low as 103 magnetically labeled molecules can be detected; using the force-induced remnant magnetization spectroscopy (FIRMS) technique, specific molecular bonds can be distinguished from nonspecific absorption. These progresses have paved the way for the research reported here. In this dissertation, I will present two major achievements in resolving molecular information using the FIRMS technique. The first is obtaining high resolution in measuring the noncovalent binding forces between molecules using the FIRMS technique. The force resolution of 1.8 pN is nearly an order of magnitude better than the current state-of-the-art technique—atomic force microscopy (AFM). This high resolution enables precise determination of the targeted noncovalent bonds hence the molecules of interest. DNA duplexes with a single basepair difference can be completely resolved in the same sample, which cannot be accomplished by AFM or any other force spectroscopic techniques. The other accomplishment is using acoustic radiation force to distinguish molecular interactions for the first time. Antibodies of different subclasses and DNAs with different binding forces can be clearly identified. The small size of the ultrasound transducer allows for potential integration with the atomic magnetometer. The resulting instrument will be uniquely suitable for noninvasive manipulation of molecular interactions. This dissertation is organized as follows. In Chapter 1, I will provide an overview of magnetic materials and the related detection techniques. The principle of FIRMS will be introduced. Chapter 2 will be the principle of atomic magnetometry. This is the basis of the detection method for FIRMS and its derived techniques. Chapters 3 and 4 will describe in details the research progresses, both of which have been published. In Chapter 5, I will present an ongoing project that concerns building an apparatus that integrates an ultrasound probe and an atomic magnetometer. The goal is to ultimately achieve detection and manipulation of molecule-specific noncovalent bonds. Preliminary results have demonstrated feasibility of such an apparatus.Item Direct Quantitative Detection of Microrna with a Purification-Free, Amplification-Free and Label-Free Technique(2018-05) Yang, Haopeng 1988-; Wang, Yuhong; Fox, George E.; Gunaratne, Preethi H.; Xu, ShoujunMicroRNAs play key roles in various types of cellular processes such as cell proliferation, and differentiation via post-transcriptional regulation. Therefore, they have great potential of being promising biomarkers for disease diagnosis and as a result have attracted much attention. To address the increasing need for their precise detection, many quantitative approaches have been developed. Recently, a new direct detection technique, Exchange-Induced Remnant Magnetization Spectroscopy (ExIRMS) has been reported by our laboratory with no amplification or washing procedures. However, it has not been demonstrated the bio-compatibility in the detection of cellular microRNAs and circulating microRNAs. Therefore, in this dissertation, I focus on the detection of microRNAs by ExIRMS in cells as well as body fluid. Firstly, the exchange reaction between the target microRNA and the probe with one mismatched base during competitive binding with the complementary magnetically labelled RNA was utilized. The results showed that ExIRMS was sufficiently robust to achieve a high sensitivity with 105 molecules for detecting microRNAs in cell lysate as well as clinical serum. Neither purification nor amplification are required for detection, which maximally simplify the analysis process and avoid possible loss during sample preparation for absolute quantification. Additionally, this technique was compared with conventional method, and it is confirmed that ExIRMS is comparable to qPCR for purified RNA from both cells and sera. Moreover, accurate absolute quantification of circulating microRNAs requires serum with fractionation and appropriate treatment such as proteinase K and Triton X100 was demonstrated. Further, ExIRMS was evaluated and optimized for specificity and sensitivity. It was demonstrated that new mismatch (G-U) at position 19 is able to distinguish let-7a from other paralogs of let-7 while old mismatch (C-C) can detect all let-7a family in an energy-based preference. By titration, it was also determined that 1 pM of mismatched-strand with 6 pM of let-7a complement plus 250 thousands of beads was optimal for the platform. In addition, force spectrum was applied prior to ExIRMS to remove non-specific interaction on the surface. Together, the evaluation and optimization provided us a promising future of ExIRMS in microRNAs profiling, which can be further applied in disease diagnosis and prognosis.Item Global Structural Mechanism of Translation and Evolution of RNA towards Modern Life(2017-12) Paci, Maxim 1988-; Fox, George E.; Wang, Yuhong; Briggs, James M.; Willson, Richard C.The structure of the ribosome and its function is explored through superposition of published crystal structure data. “Pivoting elements” are discovered in the ribosome - structural weakness caused primarily by UG mismatches and similar motifs. This structural weakness allows for functionality in the ribosome and consequently all RNA. An overall connected network of motion is further revealed in the ribosome propagating from cofactor GTPases through the decoding center and back. The mobile network is highlighted by series of equally mobile ribosomal proteins. A series of questions regarding the origins of life are explored through the application of dynamic combinatorial chemistry to biochemical systems. A series of experiments were proposed, defended and performed, which show that RNA can, in an aquatic environment, gain information size and become, over time, robust towards hydrolytic stress.Item Measuring the Mechanical Forces of Elongation Factor G Mediated Translocation(2022-04-14) Gavriliuc, Miriam Nicolette; Wang, Yuhong; Briggs, James M.; Do, Loi H.; Gunaratne, Preethi H.The ribosome is the ribonucleoprotein complex that is responsible for the correct translation of mRNA into protein. This complex is associated with the GTPase elongation factor G (EF-G), which catalyzes translocation on the ribosome, however, the mechanism is poorly understood. Crystallographic studies revealed a previously unknown compact conformation of EF-G, implying large conformational changes. The biological relevance of this change was not revealed, however, because of the artificial crystal lattice present in x-ray studies. To explore this, an EF-G mutant carrying only two cysteines (M5 EF-G) was generated and internally crosslinked to itself, using two lengths of crosslinker. One that restricted EF-G from fully extending (BM(PEG)6) and one that allowed EF-G to undergo full conformational changes (BM(PEG)11). BM(PEG)11 crosslinked EF-G functioned comparably to wild type, while BM(PEG)6 crosslinked EF-G resulted in a lower force exerted. The BM(PEG)6 crosslinker also reduced ribosomal translocation efficiency to half, compared to that of the longer crosslinker and non-crosslinked EF-G. This force reduction did not result in frameshifting, confirming that EF-G does exert a force, but does not contribute to reading frame maintenance. Studying the overall conformational changes was important for understanding EF-G mechanism, however, it was also important to observe the details leading to the full conformational changes. In a second exploration of EF-G, the effector loop region was studied. The effector loop region spans approximately 7 residues within the GTPase center, the region where GTP is bound and hydrolyzed, on domain I of EF-G and contains important interactions for the function of EF-G. By introducing single site substitutions in the effector loop, the role of each residue was examined. Six variants were produced, and of the six, one was completely unable to hydrolyze GTP, while another exhibited reduced GTPase activity. Not a single of the six was able to promote translocation at a rate comparable to M5 EF-G, effectively abolishing the function of EF-G on the ribosome. By studying the effector loop of EF-G, further understanding of the roles of the residues within this region, and how they contribute to the overall function of EF-G was obtained.Item Mechanistic Study and Ligand Optimization of the Copper(I)-Catalyzed Azide-Alkyne Cycloaddition (CuAAC) Reaction(2016-12) Chen, Haoqing 1989-; Cai, Chengzhi; Daugulis, Olafs; Thummel, Randolph P.; Lubchenko, Vassiliy; Wang, YuhongThe copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction is a prime example of “click reaction” that has been widely applied in diverse fields. The mechanism of this reaction has been proposed involving di-copper complexes as the kinetically favored active species, which are difficult to detect due to the multiple fast equilibria between the copper complexes and the instability of copper(I) to disproportionation and aggregation. We first investigated the intermediates in CuAAC reaction with the widely used tris(triazolylmethyl)amine ligand. Using electrospray ionization mass spectrometry (ESI-MS), we detected unprecedented tri-copper(I) acetylide and triazolide intermediates. By linking an alkyne with the ligand, we enriched the di- and tri-copper(I) acetylides in aqueous solutions, and quantitatively analyzed the reactivity under stoichiometric and catalytic conditions. The di-copper(I) reaction mechanism was energetically preferred under stoichiometric conditions, while the tri-copper(I) intermediates are more stable and the reaction can go through both pathways under catalytic conditions. We obtained the single crystal X-ray diffraction structure of the tri-copper(I) acetylide intermediate bearing one tris(triazolylmethyl)amine ligand, which displayed high catalytic activity in CuAAC reaction. Based on the ESI-MS results and the crystal structure, we proposed the tri-copper(I) CuAAC reaction mechanism. Under stoichiometric condition, the tri-copper(I) acetylide directly coordinates with azide and generates an azide-acetylide adduct to form the triazole ring. Under catalytic condition, the reaction could involve an internal copper(I) dissociation from the acetylide to reduce the steric hindrance. The activation energy of the proposed di-copper(I) pathway was calculated 2 kcal/mol lower than the tri-copper(I) pathway. To study the ligand effect on the oxidative side reaction of CuAAC that caused significant damage to biomolecules, we synthesized a series of tripodal amine ligands bearing triazole or phenyl substitution groups, which can coordinate to copper(I) with five- or six-membered chelating arm length. The best copper(I) ligand in CuAAC reaction were screened. In addition, we designed a platform for high throughput screening of copper(I) catalyst exhibiting high CuAAC activity and high stability. The lead catalyst first generating the product upon addition of a low concentration of azide to the library can be identified by ESI-MS. The feasibility of this approach was studied using the tris(triazolylmethyl)amine ligand.Item Mutagenesis of Escherichia coli Elongation Factor G for Domain IV sm-FRET and Force Studies(2023-04-13) Desales, Caycel John P.The translation of mRNA into protein is performed by the ribosome. This ribonucleoprotein complex consists of two subunits which come together to form three tRNA binding sites; the A-, P-, and E-site. The complex is associated with several proteins, one of which is Elongation Factor G (EF-G) which advances the mRNA sequence through the ribosome in a process known as translocation. During translocation, tRNAs move from the A- and P-sites to the P- and E-sites, respectively, and the ribosome slides the mRNA exactly three nucleotides. Two key residues (Q508 and H584) in the domain IV region of EF-G form interactions with nucleotides of the A-site tRNA during translocation. Previous mutation attempts using Q5 hot-start polymerase for two substitutions at this site (Q508A and H584A) resulted in failure. Low plasmid yield, undesired off-site mutations, and high polymerase chain reaction (PCR) failure rates were some of the primary issues faced when using this enzyme. In this experiment, a new polymerase enzyme was tested to determine if the desired mutations can be successfully amplified via PCR. Gel electrophoresis was used to confirm the presence of recombinant plasmid following PCR. Plasmid was isolated via midi-prep, and sequencing for the H584A mutant was performed using Sanger sequencing. These findings suggest that the H584A substitution mutation was not successfully inserted into Escherichia coli pDNA due to a deletion of G1742, causing a -1 frameshift. This project will be re-attempted using PAGE purified primers and a new PCR kit with fresh polymerase.Item MUTAGENIC EFFECTS OF CHROMIUM (IV) ON HUMAN ENDOTHELIAL AND LUNG EPITHELIAL CELLS(2014-08) Wang, Mu 1988-; Bose, Rathindra N.; Gilbertson, Scott R.; Thummel, Randolph P.; Wang, YuhongEffects of a chromium (IV) compound,diperoxoaquoethylenediaminechromium (IV) monohydrate (Cr(IV)-DPO), on human umbilical vein endothelial cells (HUVEC) and human bronchial epithelial cells (BEAS-2B) were investigated through morphological, cell viability, gene expression, and cDNA sequencing assays. The cell morphology showed both HUVEC and BEAS-2B cells suffered significant but different degrees of damage and demonstrated features indicating carcinogenesis when these cell lines were exposed to Cr(IV)-DPO for different time durations. Results from viability and morphological assays indicated that genotoxic damages of Cr(IV)-DPO to HUVEC and BEAS-2B cells were both concentration and time dependent. With longer time exposure up to 72 hr or higher concentration up to 100 µM, these damages were more pronounced. To understand the specific nature of these damages, expressions of apoptotic and DNA repair genes by RT2-PCR arrays, and cDNA sequencing studies were carried out. Expressions of most apoptotic genes of HUVEC were altered; in particular, they were over-expressed due to the chronic Cr(IV)-DPO exposure (5 µM, 48 hr), indicating that Cr(IV) inhibited cellular proliferation in response to the oxidative damage. When BEAS-2B cells were treated with the same concentration of Cr(IV)-DPO, DNA repair genes displayed responses less actively as the exposure time increased. Gene sequencing experiments on Cr(IV)- treated BEAS-2B cells exhibited remarkable mutations with transverse mutations dominated the variants. These gene mutations were random and not confined to guanine bases as reported in the literature.Item SINGLE-MOLECULE LEVEL STUDY OF THE INTERACTION BETWEEN ATR AND VITAMIN B12 - CONFORMATIONAL ALLOSTERY FOR MISCELLANEOUS FUNCTION(2022-11-09) Pan, Manhua; Chen, Tai-Yen; Baldelli, Steven; Yang, Ding-Shyue; Zastrow, Melissa L.; Wang, YuhongVitamin B12 is an essential cofactor for methylmalonyl-CoA mutase (MCM) to catalyze the conversions of methylmalonyl-CoA to succinyl-CoA in the mitochondria. The Cob(I)alamin adenosyltransferase (ATR) initiates this metabolic process by synthesizing and delivering 5-deoxyadenosylcobalamin (AdoCbl) to MCM. Failure of AdoCbl delivery to MCM will eventually form the oxidative inactive species HOCbl. The homo-trimeric ATR maximally binds two AdoCbl in its subunit interface with negative cooperativity for the MCM delivery function. A recent crystallographic study further reveals that ATR may regulate its reactivity and transportability by modulating its mobile loops. Despite the valuable thermodynamic and protein structural information, how ATR binding kinetics correlate with the protein allosteric regulation and the origin of the negative cooperativity remains unknown. Further, with the presence of HOCbl, it is also unclear how ATR could preferentially interact with AdoCbl over HOCbl to maximize the transferring efficiency. This research aims to address these questions using single-molecule approaches. Quantitatively measuring the binding/unbinding process is needed to address these questions. By developing a FRET-based single-molecule relative fluorescence (SRF) approach with the single-molecule interaction simulation (SMIS), we probe the interactions between ATR with two B12 derivatives, AdoCbl and HOCbl. The SRF trajectories capture the interactions and report the microscopic interacting dwell-time distributions, which quantify the binding and unbinding kinetic parameters and reveal a two-step binding mechanism. SMIS simulates and gives the expected kinetic characteristics based on the assigned kinetic model, and it can also be used to dissect the interaction kinetics from single-molecule FRET trajectories. Our data indicate that ATR undergoes a conformational change before binding substrates. Such conformational change slows down the second substrate binding. This negative cooperativity favors the AdoCbl release or delivery, which benefits the AdoCbl synthesis by emptying a reaction site. The comparative analysis of interaction kinetics between AdoCbl and HOCbl also informs that the apo-ATR preferentially binds AdoCbl through a conformational sampling mechanism. These findings illustrated important molecular dynamic insight into how the dynamic ATR-AdoCbl interaction mediates AdoCbl transferring. The developed SRF assay also offers a possibility to investigate protein-substrate interactions with relevant time resolutions, and SMIS enables the precise quantification of the interaction kinetic rate constants without using the traditional single-molecule analytical solution.Item Single-Molecule Quantification Methods To Detect Protein Oligomeric Stoichiometry In Cell(2023-08) Chen, Huanhuan; Chen, Tai-Yen; Baldelli, Steven; Yang, Ding-Shyue; Zastrow, Melissa L.; Wang, YuhongProtein oligomerization plays a critical role in many biological processes, but studying its quantification in cells has been challenging. In this dissertation, we developed three intracellular single-molecule methodologies based on super-resolution fluorescence microscopy to probe the oligomeric stoichiometry of cytosolic and membrane proteins. Using these techniques, we investigated the oligomeric modification of Cu/Zn superoxide dismutase (SOD1) under different conditions. We found that treating cells with hydrogen peroxide (H2O2) promotes SOD1 dimerization and decreases cellular viscosity. Additionally, we observed that SOD1 tends to monomerize under reduced and Cu-depleted conditions. Furthermore, we developed a location-based assay, probability of neighbor density (PND), which can quantify protein oligomeric stoichiometry up to trimer. Our studies provide new insights into the molecular mechanisms underlying protein oligomerization and highlight the potential of these methodologies for future research in this field. In the first chapter, we developed a single-molecule fluorescence anisotropy (smFA) assay to study the oligomeric modification of SOD1 in response to H2O2. The smFA assay was validated using monomeric and dimeric mEos4b, and then it was performed in live COS7 cells overexpressing SOD1-mEos4b. We found that treating cells with H2O2 promoted SOD1 dimerization and reduced cellular viscosity in 2 h. However, after 24 h, the cells returned to a steady state similar to the basal state. We further developed a single-molecule Förster resonance energy transfer (smFRET) assay to quantitatively investigate the oligomeric state of SOD1 under different conditions. Firstly, we observed that 70% of SOD1 existed as dimer under basal condition. Interestingly, SOD1 tended to monomerize under both reduced and Cu-depleted conditions. However, under Cu-stressed conditions, no SOD1 monomer was detected. Additionally, under oxidative conditions, the SOD1 monomer component decreased to approximately 20%. In the last chapter, we introduced the PND assay, which links spatial information with protein oligomeric stoichiometry, up to the trimer level. Firstly, we developed a theoretical description of PND for pure oligomers, which was validated using simulated ground truth data. Since proteins can exist in mixed oligomeric states, we successfully extended the theoretical model to account for mixed stoichiometry. Furthermore, we demonstrated the assay's ability to accurately quantify protein levels with negligible error.Item Super-Resolution Single-Molecule Fluorescence Imaging for Studying Intracellular Transition-Metal Catalysis & Reductive Amination of Carbonyl Compounds Catalyzed by Half-Sandwich Iridium Complexes Under Mild Reaction Conditions(2022-04-21) Nguyen, Dat Phat; Do, Loi H.; Miljanić, Ognjen Š.; Chen, Tai-Yen; Lee, T. Randall; Wang, YuhongIn recent years, the field of intracellular catalysis has gained much popularity due to the ability of transition-metal complexes to promote selective reactions inside living organisms. Unfortunately, precise quantification of intracellular activity of a metal catalyst cannot be achieved using current ensemble-averaged methods. Instead, we proposed that single-molecule measurement techniques might provide valuable kinetic and mechanistic data that would enhance our understanding of intracellular catalytic behavior. Super-resolution fluorescence microscopy is an advanced optical technique that allows scientists to visualize individual biomolecules inside living cells. This method has been employed to observe and characterize various biological events at a molecular level. We herein describe our work using single-molecule imaging to evaluate the ruthenium-catalyzed uncaging of alloc-protected fluorescent probes inside living cells. This study will allow us to derive important kinetic and biological insights into the intracellular protecting group cleavage process. In the second project, we demonstrated that the conversion of aldehydes and ketones to primary amines could be promoted by half-sandwich iridium complexes using ammonium formate as both the nitrogen and hydride source. The reductive amination of a variety of carbonyl substrates in common polar solvents at 37 °C provided excellent selectivity for the amine over alcohol product. In aqueous media, selective reduction of carbonyls to primary amines was achieved in the absence of acids. Unfortunately, at catalyst concentrations of <1 mM in water, reductive amination efficiency dropped significantly, which suggests that this catalytic methodology might be not suitable for aqueous applications where very low catalyst concentration is required (e.g., inside living cells).Item The Mechanism of Ribosomal Translocation Revealed by Single-molecule FRET and Super-resolution Force Spectroscopy (SURFS)(2020-12) Lin, Ran; Wang, Yuhong; Fox, George E.; Fujita, Masaya; Cai, ChengzhiDuring protein synthesis, peptidyl tRNA at the A site moves to the P site, and deacylated tRNA at the P site moves to the E site. This step in protein translation is called translocation and is catalyzed by a conserved GTPase--elongation factor G (EF-G). This dissertation addresses the role of EF-G and EF-G-induced GTP hydrolysis in the typical translocation event. In addition, we evaluate the role of EF-G in abnormal translocation events like ribosomal stalling and frameshifting during the translation process. The rate of tRNA translocation during protein translation is accelerated 50-fold by the EF-G-induced GTP hydrolysis. It is not clear that the GTP hydrolysis causes some conformational changes in EF-G or powers tRNAs translocation directly. To achieve our experimental objectives, we developed a dual FRET system to track the internal interaction of two EF-G domains while simultaneously tracking the interaction of ribosomes and tRNAs. The FRET data shows that GTP hydrolysis and ribosome are required for EF-G conformational change but are not coupled directly with tRNA translocation. Meanwhile, translocation can be paused when the ribosome stalls on an mRNA encoding a polyproline sequence. One possible reason for ribosomal stalling is the slow peptide bond formation between prolines. The FRET system was used to track sensitive changes in the interaction of tRNAs and ribosomes to detect the kinetics of the prolyl peptide bond formation. Our observations suggest that ribosomal stalling is caused by the destabilization of A-site tRNAs in the cases where several consecutive proline codons enter the mRNA decoding center. Furthermore, translocation can also be misled by ribosomal frameshifting. This process is influenced by both the mRNA sequence and secondary mRNA structure. Super-resolution force spectroscopy (SURFS) was used to determine the force in the process of ribosomal translocation precisely. Neomycin was applied to trap different phases of ribosomal frameshifting. Surprisingly, four stages were observed when studying the movements at the 5'-end of mRNA by SURFS. It was found that during mRNA translation, its 5' terminus is always more motile than the 3' terminus. It was concluded that EF-G rescues the frameshifting by stabilizing the ribosome with compromised conformational changes.