Single-molecule spectroscopy: investigations of protein folding to multi-laboratory consistencies on proteins

The investigation of complex biological processes has been challenging and require a variety of sophisticated tools to interpret the underlying processes. The study of the folding process in proteins is one of the focuses of this thesis work. To this end, both spontaneous and chaperone- assisted folding mechanisms were investigated. Single-molecule fluorescence spectroscopy has been extensively applied to the study of biomolecular bindings, conformational changes, and their dynamics due to its high sensitivity, time resolution, and its ability to differentiate between homogenous and heterogenous populations. Specifically, single-molecule Förster Resonance Energy Transfer (smFRET) studies on protein folding have elucidated the basic mechanisms of spontaneous protein folding, and properties of the chaperone-substrate interactions. The possibility to measure at low concentrations making it possible to avoid the aggregation, which is difficult to avoid in ensemble experiments. To investigate the spontaneous folding mechanisms in large multi-domain proteins, two-color smFRET studies were carried out on a slowly folding version of the two-domain Maltose- binding protein (MBP). Three-color smFRET, an extension of typical two-color smFRET to three-colors, was applied on specifically labeled MBP to visualize the co-ordination between the domains as they fold. Chaperone-substrate interactions are crucial to process the substrates and thus enable them to carry out their physiological function. Cavity confinement effect of GroEL/ES, a bacterial Hsp60 on MBP folding landscape was demonstrated. Another substrate protein, p53-DNA-binding domain was probed concerning the combined action of Hsp70 and Hsp90 chaperone on its folding. To conclude the thesis work, a smFRET comparison study on proteins involving 16 laboratories was undertaken to assess the accuracy and precision of smFRET measurements as well as to determine a detection limit for dynamic motions in proteins

STRUCTURAL STUDIES OF INTERFERON REGULATORY FACTOR 4: A MOLECULAR PERSPECTIVE OF ITS REGULATORY MECHANISM

Interferon (IFN) regulatory factor family member 4 (IRF4) is a transcription factor that serves specific roles in transcriptional regulation of IFN responsive genes and is indispensable in B- & T-cell differentiation. IRF4 like the other members of the family has two major domains- the N-terminal DNA binding domain (DBD) essential for its recognition and binding to the Interferon Stimulated Response Element DNA sequence and a C-terminal Interferon activation domain (IAD) thought to maintain IRF4 in an auto-inhibited inactive state and is also critical in its activation. A putative unstructured linker connects the DBD and IAD. Activation in most members of the IRF family requires phosphorylation to induce homo and hetero-dimerization. In contrast, IRF4 functions primarily through ternary complex formation involving different proteins including PU.1 and MyD88. The IRF4IAD has a C-terminal auto-inhibitory region (AIR) that has been proposed to physically impede the DBD from interacting with DNA in the absence of its binding partner. To understand the activation mechanism in molecular detail we determined the crystal structure of the IAD of IRF4 and also performed small-angle X-ray scattering (SAXS) studies. Our data reveals that the surface electrostatics of IAD and presence of additional loops confers exclusivity to IRF4 in the IRF family. SAXS studies suggest that the AIR is structured and makes interactions with the putative linker. We also performed analytical ultracentrifugation studies, fluorescence anisotropy binding experiments and SAXS studies on full-length IRF4 as well as on constructs where the first 20 residues, exclusive to IRF4 or the AIR were removed. We observe that the first 20 residues are critical in decreasing the binding affinity of full-length IRF4 to DNA. In addition, the putative linker of IRF4 connecting the N- and C-termini appears to be a folded domain and interacts with AIR. Also, overall full-length IRF4 appears as an elongated molecule and the N- and the C-terminal domains are arranged on either ends of full-length IRF4. Moreover, there are no signs of huge conformational changes in the protein during the activation process. Taken together, based on our data we propose that there is no auto-inhibited state for IRF4. Furthermore, it is the binding affinity of full-length IRF4 that is increased in the presence of its binding partner most likely through modest conformational changes

Applications of Atmospheric Pressure Plasma in Surface Engineering

Plasma processing of materials has grown to be a key technology for various industrial applications. Low pressure plasmas have found wide applications; they require expensive vacuum systems and need orderly maintenance. Atmospheric pressure plasma jets (APPJs) on the other hand are less technically demanding. APPJs can generate a high flux of active species and are a promising alternative to low pressure plasmas for surface treatment. For an APPJ the plasma is not confined within the dimensions of the electrodes and can be directed towards the desired region. This dissertation is aimed at three novel applications of atmospheric pressure plasmas: printing nanomaterials, functionalization of nanomaterials and deactivation of airborne microbes. For all the contributions presented in this thesis emphasis have be given on studying the effects of plasma on surfaces. A novel APPJ based printing technique is proposed and developed to address issues of material degradation in conventional printing techniques. The process involves printing using nano-colloidal ink. The novelty of this printing technique is that it can tune the electronic properties of the nanomaterials in-situ while printing. Near edge X-ray fine structure (NEXAFS) spectroscopy of the deposited copper nanoparticles confirmed that the oxidation state of copper can be reduced equally at the surface and in the bulk. Also, by varying the gas mixture in the plasma the morphology of the films can be varied from uniform to porous. The film formed from copper nanoparticles tend to be insulating but can be transformed to conductive films through use of APPJ processes. Graphene oxide (GO) has found applications in multi-junction devices as charge transport layers or transparent electrodes. This is because, the work function of GO can be tuned to the device specifications. A lower power APPJ has been used to dope GO films with nitrogen. High resolution X-ray photoelectron (XPS) and NEXAFS spectroscopy revealed that the plasma induces finite changes in the surface chemistry and also influences the electronic properties of GO. Kelvin probe microscopy on the functionalized films has shown that the work function of GO can be tuned by 120 mV. This variation has been linked to the specific nitrogen configuration in the graphitic lattice. An APPJ device has also been used for depositing graphene oxide (GO) films. Hummer’s method is widely used for the oxidative exfoliation of graphite. Due to the use of strong acids, the resultant GO suspension is highly acidic and need extensive dilution to neutralize pH. It has been demonstrated for the first time that an APPJ can in-situ reduce highly acidic graphene oxide while deposition. XPS and NEXAFS spectroscopy revealed marked differences in the oxygen containing functional groups after deposition. Both NEXAFS and Raman spectroscopy revealed the healing of sp2 graphitic structure. Subsequent increase in conductivity was also observed from the electrostatic force microscopy measurements. The decontamination of airborne microbes using an atmospheric pressure dielectric barrier discharge has been demonstrated. Here, air has been used as the process gas and is found to be highly efficient in the inactivation of microbes. After interaction with the plasma, the physical structure of the microbes was found to be severely distorted and changes in surface chemical composition were also observed from NEXAFS studies. These physiochemical changes lead to the annihilation of microbes

Role of the F-BAR Protein Hof1 in the Regulation of Chitin Synthesis and Cytokinesis in Yeast

Remodeling of the plasma membrane and extracellular matrix (ECM) at discrete cellular locations plays important roles in various cellular processes including angiogenesis and cytokinesis. In the budding yeast Saccharomyces cerevisiae , membrane trafficking delivers enzymes essential for the synthesis of the cell-wall (yeast ECM) component chitin to the bud neck at different phases of the cell cycle. During early stages of budding, a Chs3-synthesized chitin ring is deposited at the base of the new bud that is required for bud-neck integrity and normal cell shape. During cytokinesis, actomyosin ring contraction is linked to the formation of a Chs2-synthesized chitinous disk to divide the mother and daughter cells called the primary septum. Chs3-synthesized chitin also plays an auxiliary rote to Chs2 during cytokinesis. Here, I show that the F-BAR protein Hof1 is involved in the endocytic removal of Chs3 from the bud neck alter chitin ring deposition and possibly later after cytokinesis. I also discuss work to show that Hof1 is involved in the localization and function of Inn1, a C2-domain containing protein essential for synthesis of the primary septum during cytokinesis

What Controls the Controller: Structure and Function Characterizations of Transcription Factor PU.1 Uncover Its Regulatory Mechanism

The ETS family transcription factor PU.1/Spi-1 is a master regulator of the self-renewal of hematopoietic stem cells and their differentiation along both major lymphoid and myeloid branches. PU.1 activity is determined in a dosage-dependent manner as a function of both its expression and real-time regulation at the DNA level. While control of PU.1 expression is well established, the molecular mechanisms of its real-time regulation remain elusive. Our work is focused on discovering a complete regulatory mechanism that governs the molecular interactions of PU.1. Structurally, PU.1 exhibits a classic transcription factor architecture in which intrinsically disordered regions (IDR), consisting of 66% of its primary structure, are tethered to a well-structured DNA binding domain. The transcriptionally active form of PU.1 is a monomer that binds target DNA sites as a 1:1 complex. Our investigations show that IDRs of PU.1 reciprocally control two separate inactive dimeric forms, with and without DNA. At high concentrations, PU.1 forms a non-canonical 2:1 complex at a single DNA specific site. In the absence of DNA, PU.1 also forms a dimer, but it is incompatible with DNA binding. The DNA-free PU.1 dimer is further promoted by phosphomimetic mutants of IDR residues that are phosphorylated in B-lymphocytic activation. These results lead us to postulate a model of real-time PU.1 regulation, unknown in the ETS family, where independent dimeric forms antagonize each other to control the dosage of active PU.1 monomer at its target DNA sites. To demonstrate the biological relevance of our model, cellular assays probing PU.1-specific reporters and native target genes show that PU.1 transactivation exhibits a distinct dose response consistent with negative feedback. In summary, we have established the first model for the general real-time regulation of PU.1 at the DNA/protein level, without the need for recruiting specific binding partners. These novel interactions present potential therapeutic targets for correcting de-regulated PU.1 dosage in hematologic disorders, including leukemia, lymphoma, and myeloma

Estrogen and Progesterone enhance Neisseria gonorrhoeae Transmigration across a Polarized Monolayer via a Mechanism that Hijacks EGFR

Gonorrhea, a common sexually transmitted infection, is caused by the gram-negative bacterium Neisseria gonorrhoeae. In the female reproductive tract, gonococci (GC) initiate infection at the apical surface of columnar endocervical epithelial cells. These cells provide a physical barrier against mucosal pathogens by forming continuous apical junctional complexes between neighboring cells. This study examines the interaction of GC with polarized epithelial cells. We show that viable, but not gentamicin killed, GC preferentially localize at the apical side of the cell-cell junction in polarized endometrial and colonic epithelial cells, HEC-1-B and T84, respectively. In GC infected epithelial cells, continuous apical junctional complexes are disrupted, and the junction-associated protein β-catenin is redistributed from the apical junction to the cytoplasm and to GC adherent sites. However, GC inoculation does not change the overall cellular level of junctional proteins. This redistribution of junctional proteins is associated with a decrease in the apical junction's barrier function against the lateral movement between the apical and basolateral membranes, but not against the permeability through the paracellular space. Disruption of the apical junction by removing calcium increases GC transmigration across the epithelial monolayer. GC inoculation induces the phosphorylation of both epidermal growth factor receptor (EGFR) and β-catenin, while inhibition of EGFR kinase significantly reduces both GC-induced β-catenin redistribution and GC transmigration. These results suggest a relationship between junction protein redistribution from the plasma membrane with the resultant weakening of the junctional complex, and an increase in the ability of GC to transmigrate. The presence of the female sex hormones estrogen and progesterone, lead to an increased degree of disruption of the junctional complex and enhance GC transmigration across the monolayer. Therefore, GC are capable of weakening the apical junction and the polarity of epithelial cells via activating EGFR, which facilitates GC transmigration across the epithelium

Characterization and function of two G-protein regulators, vertebrate LGN and Drosophila RapGAP

Ph.DDOCTOR OF PHILOSOPH

Kinetic and Conformational Characterization of Transcriptional Activator-Coactivator Interactions.

Kinetic and Conformational Characterization of Transcriptional Activator-Coactivator Interactions Initiation of transcription is achieved through a series of coupled binding equilibria commenced by interactions between DNA-bound transcriptional activators and coactivators. There is great need to understand the mechanism of these activator-coactivator interactions and design artificial transcriptional regulators as probes or potential therapeutics. However, the key mechanistic features responsible for the differential transcriptional output of these activators are yet to be well-defined. The focus of this dissertation work has been to dissect the kinetic and structural characteristics of transcriptional activator-coactivator interactions and examine the effects of small molecule modulators on these interactions. Utilizing fluorescence stopped-flow, we measured the transient-state kinetics of the transcriptional activation domains (TADs) of the activators Gal4, Gcn4 and VP16 in their DNA-bound forms binding to the coactivator Med15. We determined that they interact through the same two-step binding mechanism: an initial rapid bimolecular association step followed by a slower conformational change step. Additional analysis suggests that the tendency for an activator to undergo conformational change correlates with both its overall affinity to the coactivator and its transcriptional activity in vivo. This mechanistic study of activator-coactivator interactions was further applied to the more conformationally defined system of TADs (MLL and pKID) binding cooperatively to the coactivator KIX. The study showed that both TADs bind to KIX through a two-step mechanism similar to that of TADs binding to Med15. A small molecule fragment 1-10 from a Tethering screen covalently tethers to a cysteine mutant of the coactivator KIX domain of CBP at the MLL binding site. The additional stabilizing effect of 1-10 tethering to KIX enabled me to obtain a crystal structure of 1-10—KIX L664C. Additionally, I found that 1-10 elicits varying allosteric effects on the opposite pKID binding site of KIX, depending on the site at which it tethers. I used 1-10 tethered at different cysteine mutations as well as the MLL peptide as probes to study the allosteric effects of KIX’s pKID binding through transient-state kinetics. The results suggest that the dissociation rate constant koff between pKID and KIX correlate with their overall binding affinity KD.PHDChemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/100048/1/nkwang_1.pd