LEE- Moleküler Biyoloji-Genetik ve Biyoteknoloji-Doktora
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ÖgeBiointerfacial cell/protein–polymer interactions investigated by quartz crystal microbalance with dissipation(Graduate School, 2023-08-01) Sert Özdabak, Ayşe Buse ; Kılıç, Abdülhalim ; 521162101 ; Molecular Biology-Genetics and BiotechnologyUnderstanding cell-surface interactions is required for the development of novel and functional biomaterials. The biocompatibility and in vivo performance of these biomaterials heavily depend on processes such as protein adsorption and subsequent cell adhesion on the material surface. Common experimental approaches used to assess these processes typically involve end-point assays. However, these assays often require cell fixation or disruption and pre-or post-labeling of the cells, potentially affecting cell physiology and leading to the loss of valuable information. Furthermore, these methods cannot distinguish interfacial interactions occurring at nanometer scales between cells and the surface. The physicochemical properties of the material surface also significantly impact the performance of potential biomaterials. The interactions taking place at the interface between cells/proteins and materials are intricate and must be comprehensively understood and carefully designed to meet specific application requirements. In this context, Quartz Crystal Microbalance with Dissipation (QCM-D) emerges as an alternative and complementary method. QCM-D serves as a powerful, noninvasive technique that enables real-time and label-free monitoring of cell-surface interactions at the nanoscale. QCM-D provides distinct data regarding specific interactions at the cell - material interface, thereby offering new insights into the cell adhesion / protein adsorption behaviors. The aim of the thesis is to investigate cell-polymer interactions and to monitor the entire process in real-time using QCM-D system. For this purpose, two commonly employed polymers in the biomaterials field, Polycaprolactone (PCL) and Chitosan (CH), as well as their blends (75:25 and 25:75), were employed to investigate real-time cell adhesion behavior. As surface topography, chemical composition and wettability have significantly influence on cell adhesion process, it is important to analyze cell adhesion on well-characterized surfaces. Two types of cell lines (hFOB and 3T3) were employed to monitor cell interactions. Complementary cell culture assays were also conducted to validate the outcomes obtained from QCM-D. In the first part of the thesis, the preparation and characterization of thin films on silicon substrates and silica sensor surfaces were completed. In order to achieve homogeneous films, various parameters were investigated, i.e., polymer ratio, solvent type, substrate surface characteristics (activated with oxygen plasma or hydrophobic treatment), polymer molecular weight, and polymer ratio. The homogeneity of the films was assessed using Atomic Force Microscopy (AFM). It was founded that blends prepared with a constant amount of chitosan yielded homogeneous coatings on the silicon substrate. The chemical composition of the constructed surface was further analyzed using Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR). The spectra exhibited distinct peaks at 1725 cm-1 for PCL and 1645 cm-1 and 1584 cm-1 for chitosan, confirming the successful coating of both polymers onto the surface. Analysis of AFM images over a scanned area of 100 µm2 revealed that pure polymers produced morphologically homogeneous films, whereas the blend surfaces displayed visible domains. Particularly, 75:25 PCL/CH blend exhibited micrometer-scale domains. Comparatively, thin films prepared with pure polymers displayed smoother surfaces compared to the blends. In contrast, the blends prepared with a ratio of 75:25 exhibited the highest roughness value. In terms of zeta potential, pure PCL films exhibited the highest negative value (-88 mV), whereas pure chitosan films displayed the lowest negative value (-15 mV) at pH 7.4. Blend film zeta potential values were between these two extremes. Film thickness analysis revealed that pure chitosan films had the smallest thickness (ca. 10 nm), whereas 75:25 PCL/CH blend film had the greatest thickness (ca. 55 nm). The pure PCL film and the 25:75 blend film had thicknesses of approximately 38 nm and 14 nm, respectively. In addition, in situ spectroscopic ellipsometry was employed to determine swollen polymer thicknesses. It was observed that PCL, being a hydrophobic polymer, did not swell much in aqueous solutions. Chitosan thin films exhibited the highest degree of swelling. The blend films exhibited swelling degrees between those of the pure polymer films, while higher PCL amount (75:25) resulted in reduced swelling, as expected. Before the cell adhesion studies, protein adsorption studies onto the constructed films was conducted. Bovine Serum Albumin (BSA) adsorption was monitored in real-time using both QCM-D and spectroscopic ellipsometry at various pH values at room temperature. In the case of pure PCL film, BSA adsorption onto pure PCL film showed a consistent frequency change upon adsorption with QCM-D for all pH values investigated. However, for the other investigated films, the presence of chitosan led to pH dependent adsorption behavior. At pH 4.5, both BSA and CH were positively charged, resulting in adsorption under repulsive conditions. At pH 6.0, the electrostatic attraction between the polymer chains and BSA led to higher adsorption on films containing chitosan. The lowest frequency decrease, i.e., mass load, was observed at pH 7.4 compared to pH 4.5 and 6.0. These findings indicate that blend composition, pH and ion presence in the environment have a substantial influence on protein adsorption. To compare the adsorbed protein amounts determined by QCM-D and ellipsometry methods, diverse models were applied. When two methods are assessed, the protein quantity derived from QCM-D data was consistently higher than that obtained by ellipsometry. The amount of protein calculated from ellipsometric data was similar for the blend films and pure chitosan films for all pH values investigated. However, higher values were evident in QCM-D method due to the inclusion of coupled water in the calculations. In addition, fibrinogen adsorption presented composition dependent behavior on thin films. The highest adsorbed fibrinogen amount was monitored on pure PCL films. In contrast, no significant protein adsorption was monitored on pure chitosan films. Consequently, the adsorbed amount of fibrinogen decreased with an increasing percentage of chitosan in the films, which predominantly showed an inverse correlation with the surface hydrophilicity. Following the comprehensive characterization of the films and conduction of the protein adsorption experiments, the cell adhesion behavior of two cell lines, human fetal osteoblastic (hFOB) and mouse fibroblast (NIH/3T3), was monitored on constructed films using QCM-D. For this purpose, the cells were introduced into the QCM-D chamber and allowed to flow for 1 hour. Initial cell sedimentation after 1 h resulted in reduced cell deposition as the chitosan ratio increased in the film. This trend was consistent for the both cell lines in the first hour. Subsequently, changes in frequency and dissipation were monitored over an 18-hour period. Complementary cell culture assays were performed to validate the observations of QCM-D. For this purpose, fluorescence images and live cell images at various time intervals were captured. Distinct QCM-D signal patterns were found for the investigated cell lines, indicating the influence of the varying interfacial properties on cell adhesion, which is also dependent on the specific cell type. In the case of hFOB cells, fully spreading was observed on pure PCL films, with elongated morphologies as confirmed by fluorescence microscopy and scanning electron microscopy (SEM). Corresponding QCM-D signals showed the highest frequency drop and the highest dissipation. Blend films supported hFOB cell adhesion, but with lower dissipation values compared to the PCL film. This might be attributed to higher rigidity at the hFOB cell−blend interface, because these cells did not progress to the further stages of spreading after secretion of their extracellular matrix (ECM) proteins. Variations in the QCM-D data obtained from the blend films could be attributed to differences in the morphology of the films. Pure chitosan films showed limited hFOB cell adhesion, accompanied by low frequency drop and low dissipation. The initial sedimentation of 3T3 cells onto the constructed surfaces similarly showed dependence on the surface composition. Unlike the behavior of hFOB cells, 3T3 cell lines did not adhere to pure chitosan surfaces, evident from consistent positive frequency signals. The highest frequency change was observed on reference silica surface, with dissipation gradually decreasing. This behavior indicated an average number of cells remaining in ECM remodeling stage. The ΔD signal shape was similar for 75:25 PCL/CH blend to the reference silica surface, however a slight decrease in the frequency was observed after 10 h. This suggests the stronger attachment to the surface while cells lacked further spreading stages on 75:25 PCL/CH blend. 3T3 cells on 25:75 PCL/CH blend showed substantial frequency drop after 10 h, which accompanied by an increase in dissipation. This behavior corresponded to the later stages of cell adhesion, implying that cells probably underwent actin remodeling and fully spreading on the surface. In conclusion, distinct QCM-D signal patterns were evident in the adhesion of hFOB and 3T3 cell lines. These distinctive patterns were attributed to the variations in the strength of cell adhesion, which are influenced by both cell type and surface chemical properties. The real-time and label-free data collected through QCM-D gave us a more profound comprehension of the dynamic adhesion behavior of the cells on constructed thin films. This knowledge and understanding holds the potential to provide valuable insights for the design of novel biomaterials tailored to diverse applications.
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ÖgeExploration of novel serine protease do-like HtrA from acigöl(Graduate School, 2023-12-06) Kılıç, Meryem Menekşe ; Karagüler, Nevin Gül ; Balcı Çelik, Nurgül ; 521112113 ; Molecular Biology-Genetics and BiotechnologyEnzymes involved in industrial biotechnological processes take place in conditions of extremely high temperature, high pH, and high salinity or when there are organic solvents that have made it necessary to discover enzymes resistant to these conditions. Microorganisms in extreme environments adapt to varying levels of stress such as very high pH, temperature, salt, and pressure. For the last 20 years, researchers have focused especially on extreme environments for the discovery of enzymes that are resistant to extreme conditions, with the hypothesis that the enzymes of microorganisms adapted to these conditions can also work under extreme conditions. In this context, microorganisms can be isolated from their environment and their enzymes can be characterized by traditional microbiological methods. Besides this, new enzymes have been discovered by the method called 'metagenomics', which is not based on culture. Environments with high salt concentration are divided into two in terms of their ionic compositions. Many high salt concentration environments were formed by the evaporation of seawater, also called 'thalassohaline'. Their salt content is similar to seawater and the pH varies from basic to slightly acidic. Environments with high salt concentrations, called 'Athalassohaline', are completely different from seawater in terms of ionic composition. Acıgöl, which is our study area, is a lake with high salt concentration, which is included in the 'Athalassohaline' state group. In this study, samples from Acıgöl were employed. Acıgöl is located between the provincial borders of Denizli and Burdur in the Aegean Region of our country. Looking at the chemical composition of the lake, it is seen that Na+, K+, Cl−, and SO4 2− ions are dominant. The salinity of the Acıgöl changes between 5.8%-13%, pH between 7.8-8.2, and temperature varies seasonally between 8 °C and 32 °C. These changing extreme conditions force the microorganisms in the lake to cellular and enzymatic adaptation. These organisms adapted to high salt concentration are called 'Halophilic' microorganisms, meaning salt-loving. Based on this information, the main subject of the study is the discovery of enzymes of halophilic microorganisms that can be used in difficult industrial processes. The primary objective of this study is to obtain new proteases, which are of industrial importance, by function-based screening of culturable microorganisms. In line with this goal, firstly, soil samples taken from Acıgöl were diluted in Nutrient Broth and spread on nutrient agar petri dishes containing 10% NaCl and 1% skim milk, and the species containing protease activity were determined. It was determined by the transparent region around the colonies that the isolate had protease activity, resulting from the breakdown of skim milk. With this screening method, halophilic species in Acıgöl, which actively produce protease, were determined. Sixmorphologically different species were determined. Twoshowed protease activity, and the species with t huge zones were chosen for further studies. In the second part of the study, the whole genome of the determined species was sequenced with the New Generation Sequencing method (Illumina HiSeq 2500 platform), and its serine proteases and other biotechnologically potential enzymes were determined. According to the sequencing results, it was determined that the entire genome of the isolated species was 4,708.499 bp (base pair) in length, had a G+C ratio of 36.66%, and had 4536 gene-coding sequences. In addition, it was revealed that 99.81% ratio similarity to Virgibacillus marismortui species according to 16S rDNA sequence similarity. The whole-genome average nucleotide identity (ANI) value was obtained as 99.44% and digital DNA-DNA hybridization was computed as 88.8%. The average amino acid identity ratio (AAI: Average Amino acid Identity) was calculated as 98.69%. In addition to genomic analyses, the isolated species was also examined phenotypically and biochemically. It was determined that the species was gram positive (Gr+), both alkaliphilic and moderately halophilic, motile, endospore-forming, and protease-producing bacterium. The isolated strain shows optimum growth at 37 °C with salinity and pH ranging from 5-10% and 6 and 9, respectively. As a result of this polyphasic analysis, it was conclueded that the isolate was a subspecies of Virgibacillus species, and it has been brought to the literature with the name Virgibacillus sp. AGTR. All genome information is stored in the NCBI database. Accession number JAJERH000000000. The last step of the study aimed to produce by recombinantly and characterize the serine protease from a new isolate. Among the four serine proteases determined by whole genome analysis, the Serine protease Do-like HtrA with the lowest sequence similarity rate and fewer studies in the literature was selected for recombinant production. The Serine protease Do-like HtrA is a member of the Trypsin-like serine protease superfamily (Tryp_SPc Superfamily) and S1-C subfamily. HtrA (high-temperature requirement A), a periplasmic heat-shock protein, it has two different functions. While it shows molecular chaperone properties at low temperatures, it shows proteolytic activity at high temperatures. The structure of this kind of protease differs slightly from other commercial and well-studied proteases. Due to these properties, it could be used specifically in the pharmaceutical industry. For the recombinant production of Serine protease Do-like HtrA, primers that contain EcoR I and SacI restriction sites were designed to be specific to the start and end sequences of the gene of interest (targeting the 5' and 3' ends). By using the genome of the isolated Virgibacillus sp. AGTR strain as a template, the target protease gene was amplified and ligated into the pET-28-a(+)expression vector. The cloned vector was inserted into E. coli BL21, E. coli C43 (DE3), and RosettaTM 2 expression cells to determine the best expression host cell. As a result of the purification study, the RosettaTM 2 cell was selected for expression. Expression studies were performed with 0.1 mM, 0.5 mM, and, 1 mM IPTG concentrations at 30 ºC and 37 ºC for up to 6 hours. The highest level of expression was achieved with 0.1 mM IPTG in 4 hours at 30 °C. Successfully expressed protease gene was purified by the His-tag method. The estimated molecular weight of the protein was 42100 Da and the isoelectric point was 4.53 which is calculated using the ExPASy program. As a result of purification, the molecular weight of the enzyme (42.1 kDa) was compatible with the predicted value, according to SDS-PAGE and Western blot tests.
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ÖgeInvestigation of cobalt resistance in Rhodobacter sphaeroides at molecular level(Graduate School, 2024-10-11) Atay, Güneş ; Çakar, Zeynep Petek ; 521142112 ; Molecular Biology – Genetics and BiotechnologyRhodobacter 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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ÖgeTargeting bag-1S/C-raf interaction for therapeutic intervention in cancer(Graduate School, 2022-06-05) Tatlı, Özge ; Doğanay Dinler, Gizem ; 521152114 ; Molecular Biology-Genetics and BiotechnologyIn this context, this study aims to map the interaction surface of the complex formed by Bag-1 and C-Raf, which was accomplished through the use of both molecular and structural techniques. For this, the three dimensional structure and domain architecture of the small isoform of Bag-1 were first examined by Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS), and the regions on Bag-1S that can accommodate small molecule binding were probed to assess its "druggability". To this end, Bag-1S was first purified from cell lysate using Ni-NTA affinity purification through the incorporated hexahistidine tag, and subsequently, the tag was cleaved with TEV protease. A subsequent Ni-NTA purification was carried out in a flow-through mode to collect Bag-1S separate from His-tagged TEV enzyme and impurities that contained neighboring histidines. The purified Bag-1S showed an apparent 33-kDa band in gel electrophoresis. Sample purity was estimated at over 90% using ImageJ analysis of SDS-PAGE gel. To monitor the deuteration level of the Bag-1S isoform, HDX-MS experiments with five time-points that ranged from 12 s to 24 h were carried out and revealed the identification of ~150 peptides of the Bag-1S with a sequence coverage of 98%. Using HDX-MS data, peptide-specific deuterium incorporation rates were projected onto the modeled structure of Bag-1S and deuterium uptake was analyzed on the Bag-1S full-length structure. BAG domain exhibited a more solvent-protected and stabilized structure compared to the UBL (ubiquitin-like) domain. While turn regions are more labile, the regions where the helical conditions exist remained unexchanged during the entire monitored time. Multiple interaction partners of the adapter protein Bag-1 engage specifically with the BAG domain. Interestingly, the interaction sites of these partners coincide with the regions that are most solvent-protected. The interaction site is supposed to be located in the solvent-protected region of the BAG domain, which is surrounded by charged and hydrophilic regions. This solvent-protected region in the BAG domain likely possesses an interaction region, revealing a potential "druggable" binding site. To further evaluate the binding stoichiometry of the Bag-1S with C-Raf, cross-linking assays were performed in the subsequent experiments. To accomplish this, C-Raf and Bag-1S proteins were affinity-purified, which was followed by the combination of purified proteins to form an in vitro complex. Covalent coupling of the formed complexes was then performed with a cross-linking agent, DSS (disuccinimidyl suberate). According to the results obtained after immunoblotting of cross-linked samples, Bag-1S and C-Raf formed a 2:2 stoichiometric complex, suggesting that Bag-1S might contribute to C-Raf activation by triggering its dimerization. After the Bag-1S/C-Raf interaction was affirmed and stoichiometrically tested, on-membrane in vitro binding experiments were conducted to selectively identify the interface of the complex. The purified C-Raf was immobilized on a PVDF membrane and incubated with purified Bag-1S in vitro. Bag-1S-bound peptides were recovered and analyzed by LC-MS/MS after the formed complex was subjected to limited tryptic digestion on the membrane. A 20-amino acid length peptide was identified as a plausible C-Raf interacting peptide in the BAG domain of Bag-1S. Further, an in silico docking study was also conducted using the protein structure of the kinase domain of C-Raf (PDB ID 5OMV) and the modeled full length protein structure of Bag-1. In some of the poses with the lowest docking energy score, K137, T140, Q144, K149, and L156 residues of Bag-1S were found to occupy the Bag-1/C-Raf binding site. This region coincides with the plausible "druggable" interaction site identified in HDX-MS and on-membrane in vitro binding experiments. Site-directed mutagenesis experiments were then carried out to confirm the identified binding interface and to evaluate if mutations in the determined peptide sequence affect the binding of Bag-1S/C-Raf or not. Upon mutagenesis, K149A and L156R substitutions significantly decreased the endogenous levels of p-C-Raf (S338) and p-MEK1/2 (217/221) in MCF-7 cells. Consistently, TAP-pull down experiments demonstrated that these substitutions impaired the interaction of Bag-1S with C-Raf, without affecting its HSP70 contact. They also led to a significant decrease in the survival of MCF-7 cells compared to wild-type Bag-1S. In addition, while these mutations did not affect the interaction of Bag-1S with its known direct interaction partners, Bcl-2 and HSP70, they resulted in the disruption of its interaction with the complexes involved in other regulatory cell survival pathways, including B-Raf, Beclin 1, and Akt. Subsequent in vitro binding experiments did not reveal a binary interaction of Bag-1S with either Beclin 1 or B-Raf, at least under our experimental conditions. Therefore, it has been hypothesized that the formation of a Bag-1/Beclin 1 or Bag-1/B-Raf complex might require the presence of C-Raf as a mediator. Further, Bag-1S interacting C-Raf region was identified by on-membrane in vitro binding experiment coupled with LC-MS/MS. Four different peptides derived from native Bag-1 and C-Raf sequences corresponding to the plausible interaction segments of the complex were designed and then synthesized by using solid-phase synthesis. The ability of the peptides to hamper the formation of a Bag-1S/C-Raf complex was tested in vitro. Of these peptides, Pep 3 that targets C-Raf binder region of Bag-1S significantly altered Bag-1S/C-Raf interaction. Pep 3 not only impeded the binary interaction of C-Raf with Bag-1S but also disrupted BAG-associated complexes of Bag-1 in TAP pull-down experiments. Inhibition of multiple Bag-1S interactions afforded by Pep 3 bolsters its potential to impair the prolonged survival of cancer cells. We therefore not only affirmed that this region on C-Raf is responsible for Bag-1 binding, but also discovered a novel peptide inhibitor targeting Bag-1S, which has the potential to be improved for cancer therapy.