The sequence-dependence of biomolecular interactions involving nucleic acids and proteins is essential for numerous processes inside the cell. Insights into the underlying molecular mechanisms have... Show moreThe sequence-dependence of biomolecular interactions involving nucleic acids and proteins is essential for numerous processes inside the cell. Insights into the underlying molecular mechanisms have been obtained using various biochemical and biophysical methods on two different levels — bulk and single-molecule. These have complemented each other as single-molecule studies excel in observing multi-state dynamic interactions, but perform only at low throughput; while bulk studies can probe many different sequences simultaneously, but providing limited kinetic information. To unite the strengths of both levels, we developed high-throughput Single-molecule Parallel Analysis for Rapid eXploration of Sequence space (SPARXS), that allows the study of molecular structure, kinetics and interactions for thousands of different sequences simultaneously at the single-molecule level. We, for the first time, combine single-molecule fluorescence with next-generation Illumina sequencing. As a proof of principle we apply SPARXS to study the sequence-dependent kinetics of the four-way DNA Holliday junction, occurring among others during homologous recombination. Using SPARXS we observe the dynamic behavior of 120,000 Holliday junction molecules covering 3750 distinct core sequences, a result unattainable with previous techniques. Overall, the mechanistic insights obtained using SPARXS will give an entirely new view on the relationship between sequence, structure and function. Show less
The research described in this thesis aims at the development of ubiquitin-based research tools to study the enzymes of the ubiquitination pathway, the ligase enzymes and the deubiquitinating... Show moreThe research described in this thesis aims at the development of ubiquitin-based research tools to study the enzymes of the ubiquitination pathway, the ligase enzymes and the deubiquitinating enzymes. These enzymes are responsible for the conjugation and the removal of the post-translational modifier ubiquitin. This small protein is involved in almost all cellular processes, and when conjugated onto a substrate protein it can signal for degradation and influence the localization, interaction, stability and activity of the protein. Therefore, the dysregulation of these processes can have detrimental effects of cell organization and survival which in turn has implications in numerous processes related to diseases. Hence, it is important to fully understand the ubiquitination pathway and how to interact with it. The ubiquitin-based research tools described in this thesis aim to shine light on parts of this pathway. Ranging from the selectivity and specificity of DUBs for specific Ub linkages in competition and the catalytic efficiency of these proteolytic cleavage processes to the selectivity and activity of ligases and the activity of DUBs in cells. All ubiquitin research tools are based on synthetic ubiquitin modified with unnatural amino acids, neutron-encoded amino acids, point mutations and/or fluorescent labels, in order to study the characteristics of the enzymes in vitro. Show less
He, J.; Wink, S.; Bont, H. de; Le Dévédec, S.; Zhang, Y.; Water, B. van de 2019
Enhanced expression and activity of protein kinases are critical in tumor cell proliferation and cancer progression. These various cancer-related kinases form intricate interdependent signaling... Show moreEnhanced expression and activity of protein kinases are critical in tumor cell proliferation and cancer progression. These various cancer-related kinases form intricate interdependent signaling networks. Evaluation of the effect of various kinase inhibitors on these networks is critical to understand kinase inhibitor efficacy in cancer therapy. The dynamic activation of some kinases can be monitored by fluorescence resonance energy transfer (FRET) biosensors with high temporal resolution. Here, we established a FRET biosensor-based high throughput imaging approach to determine ERK and AKT activity in two triple negative breast cancer (TNBC) cell lines HCC1806 and Hs578T. FRET functionality was systematically evaluated using EGF stimulation and different MEK and AKT inhibitors, respectively. Next, we assessed the effect of a kinase inhibitor library containing >350 different kinase inhibitors (KIs) on ERK and AKT kinase activity using a FRET high-throughput screening setting. Suppression of FRET-ERK activity was generally positively correlated with the proliferation phenotype against inhibitors targeting MAPK signaling in both cell lines containing FRET-ERK reporter. AKT inhibitor (AKTi) resistant HCC1806 showed decreased proliferation associated with downregulated dynamics of FRET-ERK when treated with KIs targeting protein receptor tyrosine kinase (RTK). Yet, MEK inhibitor (MEKi) resistant Hs578T showed positively correlated FRET-AKT and proliferative responses against different PI3K and AKT inhibitors. Altogether, our data demonstrate the feasibility to integrate high throughput imaging-based screening of intracellular kinase activity using FRET-based biosensors in assessing kinase specificity and possible signaling crosstalk in direct relation to therapeutic outcome. Show less
We developed a new FRET-based technique, “Fluredox”, which allows fluorescence readout of the redox state of oxido-reductases at single molecule level. Commercially available red-absorbing... Show moreWe developed a new FRET-based technique, “Fluredox”, which allows fluorescence readout of the redox state of oxido-reductases at single molecule level. Commercially available red-absorbing fluorophore ATTO655 was selected for labeling Azurin, a small blue mononuclear copper protein. Single molecule fluorescence correlation spectroscopy (FCS) of the fluorescently labeled Copper azurin in solution reveals how the position of the label in the 3-D structure of the protein affects the redox kinetics of the redox center as well as the label. Under certain redox conditions, we have been able to observe a microsecond dynamics for intramolecular ET reaction between the label and the metal center in azurin. Our results show that this FRET technique can be profitably used to study the enzyme activity of dye-labeled oxidoreductases. Show less
Proteasomes are multi-protein, multi-catalytic complexes responsible for the degradation of 80-90% of the proteins inside eukaryotic cells. Proteasomes contain a cylindrical 20S core particle (CP)... Show moreProteasomes are multi-protein, multi-catalytic complexes responsible for the degradation of 80-90% of the proteins inside eukaryotic cells. Proteasomes contain a cylindrical 20S core particle (CP) and one or two 19S regulatory particles (RP). The constitutive proteasome core particle (cCP), which is expressed in all mammalian tissues, contains three catalytically active subunits, namely β1c, β2c and β5c. Lymphoid cells express another proteasome core particle known as the immunoproteasome (iCP). In iCPs, β1c, β2c and β5c are replaced by β1i, β2i and β5i. The research described in this thesis reports on the development of new subunit-selective inhibitors and activity-based probes, on the development of an assay to simultaneously monitor all cCP and iCP catalytic activities and on the development of a method that reports on CP catalytic active subunit composition. The tools that stem from the work described in this thesis can now be used to unravel the role of each individual catalytic subunit in a chemical genetics setting (selective and (near) complete inhibition of each subunit), and to clarify the role of mCPs, in, for instance, antigen presentation and cancer. Furthermore, these tools could possibly serve as leads in the discovery of agents for future treatment of cancer and autoimmune diseases. Show less
Geurink, P.P.; Tol, B.D.M. van; Dalen, D. van; Brundel, P.J.G.; Mevissen, T.E.T.; Pruneda, J.N.; ... ; Ovaa, H. 2016
Ever since the structural data of biological macromolecules became available, there has been consistent struggle to relate this new information to the existing spectroscopy, activity and... Show moreEver since the structural data of biological macromolecules became available, there has been consistent struggle to relate this new information to the existing spectroscopy, activity and theoretical descriptions of these proteins and to understand the evolution and/or to predict the role of yet uncharacterized gene products in this light. The research presented in this thesis primarily deals with understanding the structure__function relationship of a newly discovered blue copper protein. The protein is derived from Streptomyces coelicolor and is called small laccase (SLAC). It utilizes four copper ions to catalyze the oxidation of substrate molecules concomitant with the reduction of oxygen to water. The catalytic cycle of this enzyme is studied using a variety of spectroscopic and kinetic methods in an attempt to improve our understanding of the internal operations which are critical to its functioning. The new results obtained are presented in this thesis. Show less
We study the dynamics of single molecules and individual gold nanorods in glycerol at variable temperatures. We demonstrate temperature-cycle microscopy on FRET-labeled polyproline and double... Show moreWe study the dynamics of single molecules and individual gold nanorods in glycerol at variable temperatures. We demonstrate temperature-cycle microscopy on FRET-labeled polyproline and double-stranded DNA molecules to access micro-second dynamics of single molecules, and reveal the influences of dye-dye interaction at short interdye distances on the observed FRET values. We use neutron-scattering techniques to examine the origin of solid-like structures suggested in previous reports and the influence of the thermal history. We find that crystal nucleation takes place in glycerol at temperatures very close to the glass transition temperature. This observation suggests that the thermal history of the glycerol sample needs to be controlled for studying dynamical heterogeneity in supercooled liquids. For the first time, we demonstrate gold nanorods as local viscosity reporter to study heterogeneity in supercooled liquids. Following rotational dynamics of individual gold nanorods in glycerol upon cooling below 226K, we start to observe deviations of local viscosity from the bulk viscosity of glycerol. Our observation suggests heterogeneity on relatively large length scale exists at surprisingly high temperatures. In the end, we demonstrate gold nanorods for enhancing fluorescence from single molecules and for fluorescence correlation spectroscopy at micromolar concentrations with single-molecule sensitivity. Show less
Major Histocompatibility Complex class II (MHC-II) antigen presentation is involved in the regulation of immune responses against infections and tumours and strongly linked to autoimmune diseases.... Show moreMajor Histocompatibility Complex class II (MHC-II) antigen presentation is involved in the regulation of immune responses against infections and tumours and strongly linked to autoimmune diseases. To better understand this process we performed a genome-wide RNAi screen. The effect of downregulation of each human gene on MHC class II expression and antigen loading was analyzed. Genes affecting MHC-II antigen presentation were tested for their involvement in MHC-II transcription and transport. Some genes might function in repositioning MHC-II towards the plasmamembrane upon Dendritic Cell maturation. This was confirmed by silencing these genes in immature Dendritic Cells. The novel GTPase ARF7, ARF7EP and MYO1E play a central role in this transport process. MHC-II antigen loading on intraluminal vesicles of the MHC-II compartment is facilitated by chaperone DM. In a confocal FRET study we investigated whether microdomain-forming tetraspanins CD63 and CD82, interacting with MHC-II and DM, could facilitate the MHC-II/DM interaction. MHC-II and DM preferably interacted with CD63 on the intraluminal vesicles, likely facilitating the MHC-II/DM interaction and antigen loading. MHC-II favoured interactions with CD82 on the limiting- and plasmamembrane suggesting a sorting role for CD82. This knowledge will contribute to development of new methodologies to influence MHC class II presentation in patients. Show less
Cells respond to extracellular cues via receptor signaling. In this manner, cellular behavior is under strict control of hormones, growth factors or neurotransmitters. Binding of a ligand to its... Show moreCells respond to extracellular cues via receptor signaling. In this manner, cellular behavior is under strict control of hormones, growth factors or neurotransmitters. Binding of a ligand to its cognate receptor triggers a cascade of intracellular signaling events leading to, for instance, activation of transcription factors, regulation of metabolic processes or changes in cell morphology. To regulate such __organized complexity__, molecular interactions in the cell are specific and regulated. Furthermore, these signaling pathways are strictly compartmentalized. This means that spatial restriction facilitates the coupling of a signaling component to its effectors, while it leaves other signaling compartments unaffected. Thus, compartmentalization adds to the specificity of signaling cascades. Signaling complexes are dynamically regulated, i.e. they can be assembled in response to extracellular signals. The dynamic targeting of a signaling molecule to a specialized compartment becomes manifest as a stimulus-induced translocation. In this thesis, we revealed and studied the translocations of two proteins: those of chloride intracellular channel 4 (CLIC4) and exchange protein directly activated by cAMP 1 (Epac1). Central in these studies were the microscopical techniques that allowed visualization of these translocations with maximal spatiotemporal resolution, most prominently via confocal imaging and measurement of FRET. Show less
This thesis centers around a novel fluorescence based method that allows to monitor the activity of redox enzymes and of electron (ET) or oxygen transfer proteins. It takes advantage of the fact... Show moreThis thesis centers around a novel fluorescence based method that allows to monitor the activity of redox enzymes and of electron (ET) or oxygen transfer proteins. It takes advantage of the fact that the absorption spectrum of the protein__s active site varies upon changing its redox state. This change can be translated into a change in the fluorescence intensity of a label that is covalently linked to the protein on the basis of F_rster Resonance Energy Transfer (FRET). With our method we could show that different redox proteins and enzymes can be studied down to the single molecule level. This exciting finding opens the door to the study of various redox enzymes and to monitor specific substances such as for example nitrite. Depending on the function of the enzyme under investigation a wide range of substrates can be monitored. Another example is the development of an oxygen sensor by employing proteins that are capable of binding oxygen. The findings presented in this thesis might be significant for applications in oxygen sensing and, more generally, in the fast growing field of biosensing Show less
Phosphatidylinositol 4,5-biphosphate (PIP2) has been proposed to act as a second messenger in the regulation of many cell processes. If so, then PIP2 should fulfill two important criteria's: first... Show morePhosphatidylinositol 4,5-biphosphate (PIP2) has been proposed to act as a second messenger in the regulation of many cell processes. If so, then PIP2 should fulfill two important criteria's: first PIP2 levels should vary spatially or temporally under physiological conditions, and secondly, these variations should suffice to influence cellular processes. In this thesis we have addressed the issue and provide data that support the idea that PIP2 can function as a second messenger, since PIP2 levels were found to vary significantly over time affecting cell survival, as well as cortical actin dynamics. Interestingly, these results also suggest that PIP2 influences multiple physiological processes within the same cell, apparently with spatial resolution. This view also prevails in the literature: it is widely hypothesized that the plasma membrane contains spatially confined PIP2 pools or domains. Indeed, recent studies that used GFP-tagged pleckstrin homology domains (GFP-PH) as fluorescent PIP2 sensors suggest that this lipid is enriched in membrane patches. However, we report here that this concept needs revision. Using three distinct fluorescent GFP-tagged pleckstrin homology domains, we show that highly mobile GFP-PH patches colocalize perfectly with various lipophilic membrane dyes and, hence, represent increased lipid content rather than PIP2-enriched microdomains. We show that bright patches are caused by submicroscopical folds and ruffles in the membrane that can be directly visualized with a novel numerically enhanced imaging method. Moreover, to test sub-resolutional clustering, we analyzed clustering of PIP2-binding pleckstrin homology domains by electron microscopy as well as by FRET in live cells. These assays suggest that PIP2 is neither clustered in micrometer nor in sub-resolution domains. This can be explained by the fast diffusion of PIP2 which limits the formation of small domains, as established by FRAP experiments. However, at larger scale, and especially at structures where diffusion is limiting, PIP2 gradients can be induces. Thus, our data support a model in which PIP2 has a second messenger role in the regulation of cell survival and the cortical actin cytoskeleton, but they challenge a model in which this regulation exist at a smaller scale. Show less
