LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji-Doktora
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ÖgeModeling dynein dynamics and its interactions with microtubules and microtubule-associated proteins using molecular dynamics simulations(Graduate School, 2024-10-28)Cellular transport is essential for maintaining organization within the cell, like logistics in a city, where microtubules (MTs) function as pathways connecting different areas. Motor proteins, kinesin and dynein, transport cellular cargo along these MT tracks, with kinesin mostly moving toward the plus (+) end and dynein toward the minus (−) end. Both proteins rely on ATP for energy, but they have different structures and mechanisms of action. Mutations that impair their transport can lead to neurodegenerative and developmental disorders. MT-associated proteins (MAPs) regulate this transport by interacting with MTs. In comparison, the abnormal expression of tau can cause an impairment in synaptic vesicle transport, while MAP7 assists motility of kinesin-1 while repressing those of kinesin-3. Kinesin motor proteins move cargo toward the plus (+) end of MTs (anterograde), while dynein moves cargo toward the minus (−) end (retrograde). Over 40 kinesins facilitate transport to the plus end, while a single dynein type (dynein-1) is responsible for retrograde transport. Dynein-2, however, handles intraflagellar transport in cilia. Both dyneins share a conserved structure, with ATPase activity powering their movement. Cytoplasmic dynein consists of two identical heavy chains (DHCs) and several smaller polypeptides. Each DHC has a motor domain (head) with a ring of six AAA modules and a tail domain for dimerization. ATP hydrolysis at AAA1 drives dynein motility, while the MT-binding domain (MTBD) is connected to the catalytic domain via a coiled-coil stalk. The dynein mechanochemical cycle involves four states, where ATP binding triggers MT release, and ATP hydrolysis drives the linker's movement from a bent to straight conformation, generating the force needed for dynein to step along MTs. This cycle allows dynein to transport cargo along MTs efficiently. High-resolution structures (PDB 7Z8G and 7Z8F) were used to examine the ATP-induced release of dynein from MTs. The simulations demonstrated high structural stability throughout a 3000 ns molecular dynamics (MD) simulation. The root mean square deviation (RMSD) values showed minimal deviations. Additionally, throughout the simulations, the dynein stalk and MTBD demonstrated similar angular behavior, supporting the structural integrity of dynein in its ADP-bound state. The conformational changes of dynein's linker during the priming stroke, which is one of the critical events in its mechanochemical cycle, were modeled and analyzed. To investigate the dynamics and energetics of the dynein linker, conventioal MD, steered MD (SMD), and umbrella sampling simulations were conducted on human dynein-2 in its primed state for the power stroke. The simulations demonstrated that the linker can assume a bent or semi-bent conformation, with a 5.7 kT energy barrier separating the two states. It was also shown that in the pre-powerstroke state, the linker cannot revert to its straight conformation due to steric clash with the AAA+ ring. The simulations also revealed that, when isolated from the AAA+ ring, the linker's free energy minimum is positioned near the semi-bent conformation, indicating that the linker stores energy during bending and releases it during the powerstroke. The structure of human cytoplasmic dynein-2 (PDB: 4RH7) was modified by completing missing residues and replacing ADP.Vi at the AAA1 binding site with ATP. The buttress region was cleaved and connected using a GGGG linker, followed by solvation in a water box with neutralized and ionized conditions. MD simulations were performed, totaling 4000 ns, to study the structural changes and dynamics of the cleaved dynein-2 structure. The linker angle was calculated over time, and the results showed deviation from the initial structure for one set of simulation and other three sets of simulations showed minimal deviations. The cleavage of the buttress may have induced conformational changes in the catalytic ring or the stalk, potentially shifting the linker from its bent to semi-bent state. The atomic model of MAP7 on tubulin was constructed using cryo-EM data and modeling tools like VMD and Alphafold2. A total of 4000 ns of MD simulations were performed to study MAP7's interactions with MTs. PCA was used to generate the energy landscape of MAP7, identifying four binding modes. Interaction analysis was conducted based on these binding modes. The four binding modes were revealed by PCA based clustering of MAP7 conformations. Also, intrinsically disordered C-terminal tails of MTs were interacted with the MAP7 MTBD. For Tau, a similar approach was used to model its MTBD, consisting of four repeat sequences (R1-R4). Simulations explored the interactions of Tau's MTBD with MTs, focusing on the structural integrity and dynamics of the Tau-tubulin complex over 4000 ns of MD simulations. The Tau simulations provided insights into how each repeat sequence (R1-R4) interacts with MTs, contributing to MT stability. Similar with the MAP7, intrinsically disordered C-terminal tails of MTs were interacted with the Tau MTBD.
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ÖgeInvestigation of cobalt resistance in Rhodobacter sphaeroides at molecular level(Graduate School, 2024-10-11)Rhodobacter sphaeroides, a Gram-negative α-proteobacterium, is able to perform photosynthesis and possesses a diverse array of metabolic capabilities. These capabilities include lithotrophy, respiration in both oxygen-rich and oxygen-poor environments, nitrogen-fixation, as well as the production of tetrapyrroles, chlorophylls, vitamin B12 and heme. R. sphaeroides possesses the ability to modify its metabolism, its metabolism can adapt to changes in environmental conditions and nutrient availability. This metabolic flexibility, combined with its fully sequenced genome and its ability to thrive under normal growth conditions, makes it a desirable choice for various biotechnological applications. It serves as a valuable model organism for the development and investigation of different protein expression systems. Particularly, it excels in the study of membrane protein complexes that convert light energy into electrical energy. This bacterium offers various advantages, including a fully sequenced genome, having an adaptive and well-known metabolism, and an enhanced membrane surface area when cultivated in oxygen-depleted environments. In a previous study, using evolutionary engineering, a R. sphaeroides mutant population resistant to cobalt chloride stress was obtained. This was achieved by subjecting the initial population to batch selection under gradually increased cobalt chloride stress conditions, without applying random mutagenesis prior to the selection process. Remarkably, the final mutant population exhibited resistance to cobalt levels as high as 15 mM, which has not been previously observed in R. sphaeroides. Seven mutant individuals selected from the final population were investigated, and mutant individuals were physiologically characterized. After characterization, the most resistant individual mutant (G7) with superior resistance properties was selected for more detailed analysis. Additional analysis of the G7 individual mutant from the final population revealed its ability to exhibit cross-resistance against various compounds like nickel (ІІ) chloride (2.2 mM, 2.4 mM), ethanol (8% v/v), sodium chloride (0.5 M), and aluminum chloride (5 mM), magnesium chloride (750 mM, 1M), iron (ІІ) chloride (5 mM), boric acid (30 mM, 50 mM), caffeine (20 mM) and ammonium iron (II) sulfate (5 mM). However, the underlying genomic causes of this physiological resistance capability remained unknown. The main aim of this study was to detect and analyze specific variations in the genetic makeup of the cobalt-resistant R. sphaeroides strain, known as single nucleotide polymorphisms (SNPs), which have the potential to significantly influence their resistance to cobalt chloride stress. By comprehensively exploring the interplay between these SNPs and their potential role in cobalt chloride stress resistance, this study aimed to shed light on the underlying mechanisms and pathways involved, ultimately contributing to a deeper understanding of bacterial adaptation and the development of effective strategies to combat cobalt chloride stress resistance. In this study, Flame Atomic Absorption Spectrometry (FAAS) method was used as a first step to gain insight into the cobalt resistance mechanism of G7 to determine if the mutant individual G7 retains cobalt ions inside the cell or not. As a result of the FAAS analyses, it was found that G7, which can survive in the presence of cobalt stress conditions, takes cobalt ions into/onto the cell. Moreover, to gain a detailed understanding of the underlying mechanisms behind cobalt tolerance, comparative Whole Genome Re-sequencing analysis was performed with G7 to identify and determine single nucleotide polymorphisms (SNPs) in this strain. Specifically, the G7 mutant individual and the Reference Strain (RS) were sequenced which allowed the identification of specific SNPs that potentially play a crucial role in the ability of G7 to resist cobalt chloride. By delving into the genetic variations and their potential implications, this approach aims to unravel the intricate mechanisms that contribute to cobalt resistance, thus paving the way for targeted interventions and strategies to combat this stress. According to whole genome re sequencing results, 11 missense mutations were found in various genes of the G7 mutant individual which were not present in the RS. Known mutated genes include mviN, hutC, rpoD, nifB and nhaD. Further genomic and proteomic studies would be necessary to understand the role of these genes and mutations in the cobalt chloride stress resistance of R. sphaeroides. To summarize, the comprehensive evaluation of cross-resistance tests, growth physiology observations, and genome sequencing data yielded significant insights into the genetic basis of cobalt stress resistance observed in the G7 strain. Through the identification of variations in different genes, this investigation has provided valuable information regarding the underlying mechanisms that contribute to the G7 strain's ability to withstand cobalt and other heavy metal stresses. These findings contribute to the understanding of the genetic background of heavy metal resistance and offer potential avenues for further research and targeted interventions in this field that involve R. sphaeroides.
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ÖgeInvestigation of the spastin's role in the invasion capacity of glioblastomas(Graduate School, 2024-01-05)Glioblastoma Multiforme (GBM) is the most lethal form of glioma, which are the most frequent brain tumors. Even though multimodal therapy is employed to treat GBM, tumor recurrence makes treatment almost impossible due to its robust migration/invasion potential. For the improvement of GBM therapy, it is critical to identify the proteins involved in the disease's migration/invasion process. Tumor cells require special cellular extensions controlled by cytoskeletal components to achieve migration/invasion capabilities. Spastin, a microtubule-severing protein, is mainly expressed in neurons and controls dendrites and axonal extensions of neurons. Given that the formation mechanism of these extensions in post-mitotic cells is comparable to that of specialized cell protrusions in mitotic cells, Spastin might have roles in tumor cell migration/invasion. Interestingly, Spastin has been discovered to be co-localized with actin filaments in GBM cells, suggesting that it may play a role in GBM migration ability. However, this topic has not been investigated in the literature until this study. This thesis aims to clarify the molecular mechanism underlying the shift in the intracellular localization of Spastin in GBM cells, and the potential significance of this mechanism in GBM migration/invasion ability. This study discovered for the first time that Spastin takes an active role in GBM migration. Furthermore, Spastin was discovered to interact with Pin1 via phosphorylation of Pin1 recognition motifs located in its microtubule-binding domain. Moreover, this interaction was found to direct of Spastin towards actin filaments, which promotes migration/invasion ability of GBM cells. These findings suggest that Spastin might be a therapeutic target for several tumors with a high migration/invasion capacity, like GBM.
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ÖgeMolecular dynamics studies on proteins involved in genetic variation and metabolism: DMC1 and lipase(Graduate School, 2024-12-23)Both homologous recombination and enzymatic processes, such as those catalyzed by lipases, play essential roles in biological systems, with wide-reaching applications in science and industry. Scientists have studied homologous recombination for a long time to understand its evolution, mechanics, and biological significance. The involvement of the Dmc1 protein in the context of homologous recombination is the focus of this study's thorough investigation of these features. Recombinases, such as Rad51 and Dmc1, which are found in eukaryotes, are essential for homologous recombination and DNA repair. Despite significant sequence similarities, Rad51 and Dmc1 serve different purposes; although Rad51 is essential for DNA repair, Dmc1 participates in homologous recombination during meiosis. More details about its structure and activities are revealed, including the involvement of ATP binding sites and the precise amino acids required for ssDNA binding. Loop areas have been found to be essential for DNA binding. Dmc1 has been characterized by several crystal structures showing an octameric ring configuration in the absence of ATP and DNA. Additionally, it has crystal structures in filament form. These different structures provide valuable insights into the structural flexibility of Dmc1 and its mechanism of interaction with DNA. Molecular dynamics simulations were performed on human Dmc1 protein in various oligomeric states to investigate its structural and dynamic behavior. This study aimed to explore the effects of nucleotide binding, protonation states, and peptide bond isomerization on the stability and conformational dynamics of Dmc1's N- and C-terminal domains. Key simulations included standard molecular dynamics, thermodynamic integration for pKa calculations, and umbrella sampling for free energy profiling. Protonation states of residues E162 and H295, cis-trans isomerization of the D223-S224 peptide bond, and nucleotide-binding states (ATP, ADP, or nucleotide-free) were systematically examined. Root mean square deviation (RMSD) analyses showed distinct equilibration dynamics for the C-terminal domain, while the N-terminal domain displayed significant mobility. Structural analysis revealed the connection between the protonation of E162 and its influence on DNA-binding residue R230. Besides, when E162 is protonated, the ring structure of the protein remains stable. when the D223-S224 peptide in a cis configuration, ATP binds to the Walker A and Walker B motifs in a canonical manner, similar to how ATP binding occurs in other ATPases. However, when the peptide bond is in the trans configuration, the interactions between ATP, Mg²⁺, and the Walker A and Walker B motifs are disrupted, weakening the binding. In the nucleotide-free state, the trans configuration with E162 appears more stable. Upon ATP binding, the structural behavior changes, and multiple configurations become possible. Simulations indicate that trans isomer with protonated E162, trans isomer with unprotonated E162, and cis isomer with unprotonated E162 likely exist in comparable amounts, suggesting a dynamic equilibrium driven by ATP binding and associated conformational flexibility of the protein. Additionally, molecular dynamics simulations on lipase enzymes offered comparative insights into structural flexibility and catalytic efficiency. Lipase dynamics highlighted the role of active site flexibility in substrate binding and enzymatic activity, providing a broader perspective on protein behavior. Overall, the simulations enhanced the understanding of Dmc1's dynamic behavior, interdomain interactions, and potential DNA-binding mechanisms, contributing to deeper molecular-level insights into homologous recombination processes.
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ÖgeProteomic approaches for the identification and quantification of clinically relevant biomarkers(Graduate School, 2024-10-16)Cardiovascular diseases are a significant health issue affecting people worldwide. Early diagnosis of cardiovascular diseases is crucial for the successful treatment of conditions such as heart attacks and for preventing death. Cardiac troponin I (cTnI) is a vital biomarker for the diagnosis and risk assessment of heart attacks. In healthy individuals, cTnI levels are found below 45 nanograms per liter. During and in the hours following a heart attack, it is released into the blood stream due to heart tissuedamage, leading to an increase in its levels. cTnI can be detected in the blood in various forms, including a ternary complex (cTnC-cTnT-cTnI), a binary complex (cTnI-cTnC), and free forms. It is highly susceptible to proteolysis and enzymatic changes. Consequently, various forms of cTnI, including proteolyzed, phosphorylated, oxidized, and reduced forms, can be found in the blood. All these variables lead to differences in cTnI measurements. There are many cTnI tests available on the market. The different variations circulating in the blood can be recognized by different monoclonal antibodies specific to different epitopes of cTnI. The various versions of cTnI, along with the different antibodies used, have increased the correlations between commercial tests more than tenfold, yet standardization remains challenging. Laboratories use different clinical decision thresholds depending on the test used. Different assay cutoffs have the potential to confuse physicians, leading to the misinterpretation of cTnI results; hence, there is urgency for cTnI standardization. The standardization and/or harmonization of cTnI assays is considered a high priority by the International Consortium for Harmonization of Clinical Laboratory results (ICHCLR). According to ISO 17511, the standardization of the measurement of a biomarker requires a metrological traceability chain. This chain begins with a primary reference measurement procedure (RMP), which assigns quantity values to a primary reference material (RM). Primary RMs are used to assign values to a secondary RM. With this secondary RM, values are assigned to working and product calibrators for routine quantification of the biomarker in patient samples. This traceability chain allows the values reported for patient care to be traced back to the International System of Units (SI). In this way, metrological traceability supports the long-term stability and comparability of routine laboratory measurement results. The standardization or harmonization of cTnI measurement requires the development of RMs and RMPs. The traceability chain proposed by the International Federation of Clinical Chemistry and Laboratory Medicine Working Group on Standardization of Troponin I (IFCC WG-TNI) incorporates all these standardization steps. One of the tasks that the IFCC working group is focused on to establish the proposed traceability chain is the development of a higher-order RMP. In this thesis, the focus has been on developing two different analytical methods to support the development of a RMP. Both analytical procedures involve targeted and bottom-up proteomic approaches. In both methods, isotope dilution mass spectrometry (IDMS) has been used to determine the absolute amount of cTnI. For quantifying proteins using the IDMS method, two different strategies have been employed: the protein-based calibration strategy and the peptide-based calibration strategy. Each method has its own advantages and disadvantages. The first developed analytical method allows for the determination of cTnI from human serum using a protein-based calibration strategy. In this context, human cardiac troponin complex material (NIST SRM 2921) has been selected for use as a calibrant. The troponin complex was purified from human heart tissue and consists of three subunits: troponin T (cTnT), troponin I (cTnI), and troponin C (cTnC). As an internal standard, isotopically labeled cTnI protein with the same sequence as cTnI has been used. To extract cTnI from a complex matrix like serum, an immunoaffinity enrichment strategy has been employed. As the first step of immunoaffinity enrichment, two different diameters of magnetic particles were selected: micro (Dynabeads® MyOne™, 1 μm) and nano (Nanomag®-D, 130 nm). A monoclonal antibody capable of binding to cTnI was immobilized on both types of magnetic nanoparticles, and their cTnI enrichment efficiencies were compared. Magnetic nanoparticles (Nanomag®-D, 130 nm) were chosen for further experiments because the peak areas of two selected tryptic peptides of cTnI were relatively higher. Next, the maximum loading capacity of the magnetic nanoparticles was determined. It was found that when 100 μg of antibody was added to 1 mg of particles, 59.2 ± 5.7 μg/mg of antibody could be bound. Using the synthesized nanoparticle-antibody conjugate, the required amount for cTnI enrichment from 1 ml of serum was calculated, and it was determined that 10 μl of conjugate was sufficient to capture all cTnI in 1 ml of serum for analysis. As a result of these optimizations, the isotope dilution liquid chromotograpy tandem mass spectrometry (ID-LC-MS/MS) method using the developed protein-based calibration strategy allows the measurement of cTnI in the range of 0.6 to 24 μg/L (R > 0.996). The limit of quantification (LOQ) was determined to be 1.8 μg/L, and the limit of detection (LOD) was 0.6 μg/L. Intermediate precision was found to be below 9.6%, and repeatability ranged from 2.0% to 8.7% for all quality control materials. The accuracy of the analyzed quality control materials was between 90% and 110%. Total measurement uncertainties (n=6) were found to be below 12.5% for all levels. The second developed ID-LC-MS/MS method allows the determination of cTnI in human serum using a peptide-based calibration strategy. In this method, two tryptic peptides (TLLLQIAK and NITEIADLTQK) of cTnI were selected and synthesized as calibrants. Isotopically labeled versions of the selected peptides were used as internal standards. Peptide impurity correction amino acid (PICAA) analysis was performed to assign values to the synthetic peptides, thereby producing SI-traceable primary peptide standards. Peptide-based calibration approach also employed two surrogate matrices to construct the calibration curve. The surrogate matrices were evaluated based on parameters such as linearity, accuracy, repeatability, intermediate precision, and trueness. It was observed that both matrices yielded similar results, indicating consistency in their performance. To ensure complete cleavage of the cTnI protein and enhance proteolysis yield, optimizations such as trypsin digestion methods, enzyme-to-protein ratio, and digestion time were performed. The best trypsin cleavage yield was obtained using the Filter-Aided Sample Preparation (FASP) method with a 1:10 enzyme-to-protein ratio and overnight digestion. The developed analytical method using the peptide-based calibration strategy enables the quantitative determination of cTnI in the range of 0.6–21.6 μg/L. Intermediate precision RSD was less than 28.9%, and repeatability RSD was less than 10% across all concentration levels. The recovery rate ranged between 72% and 151%. Four patient serum samples with suspected heart attack were measured using the developed method, and the results showed discrepancies of more than 50% compared to those obtained with immunoassay. Finally, the performance of the peptide-based calibration strategy was compared with the protein-based measurement strategy. In conclusion, this thesis has developed two different ID-LC-MS/MS methods using a targeted and bottom-up proteomic approaches. Both methods were compared with each other in terms of effectiveness. This efforts aim to support the metrology community in adopting new approaches and developing SI-traceable peptide and protein primary standards and/or reference procedures tailored to specific needs. Standardization and harmonization of cTnI across laboratories are undeniably complex tasks. However, the IFCC WG-TNI believes that cTnI measurement is standardizable. Given the critical role of cTnI in patient management, the significant effort invested is worthwhile. The proposed measurement methods will play a role in supporting the activities of the IFCC WG-TNI. These studies are necessary and logical steps towards the harmonization of results obtained from different test kits.
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