Moleküler Biyoteknoloji

Bu koleksiyon için kalıcı URI

https://hdl.handle.net/20.500.12846/487

Listeleniyor 1 - 20 / 90

Imprinted Polymers on the Route to Plastibodies for Biomacromolecules (MIPs), Viruses (VIPs), and Cells (CIPs)
(Springer, 2024) Yarman, Aysu; Zhang, Xiaorong; Bagheri, Mandien; El-Sherbiny, Ibrahim M.; Hassan, Rabeay Y. A.; Kurbanoğlu, Sevinç; Waffo, Armel Franklin Tadjoung; Zebger, Ingo; Karabulut, Tutku Ceren; Bier, Frank F.; Lieberzeit, Peter; Scheller, Frieder W.
Around 30% of the scientific papers published on imprinted polymers describe the recognition of proteins, nucleic acids, viruses, and cells. The straightforward synthesis from only one up to six functional monomers and the simple integration into a sensor are significant advantages as compared with enzymes or antibodies. Furthermore, they can be synthesized against toxic substances and structures of low immunogenicity and allow multi-analyte measurements via multi-template synthesis. The affinity is sufficiently high for protein biomarkers, DNA, viruses, and cells. However, the cross-reactivity of highly abundant proteins is still a challenge.
A Strep-Tag Imprinted Polymer Platform for Heterogenous Bio(electro)catalysis
(Wiley, 2024) Yarman, Aysu; Waffo, Armel F. T.; Katz, Sagie; Bernitzky, Cornelius; Kovacs, Norbert; Borrero, Paloma; Frielingsdorf, Stefan; Supala, Eszter; Dragelj, Jovan; Kurbanoğlu, Sevinç
Molecularly imprinted polymers (MIPs) are artificial receptors equipped with selective recognition sites for target molecules. One of the most promising strategies for protein MIPs relies on the exploitation of short surface-exposed protein fragments, termed epitopes, as templates to imprint binding sites in a polymer scaffold for a desired protein. However, the lack of highresolution structural data of flexible surface-exposed regions challenges the selection of suitable epitopes. Here, we addressed this drawback by developing a polyscopoletin-based MIP that recognizes recombinant proteins via imprinting of the widely used Strep-tag II affinity peptide (Strep-MIP). Electrochemistry, surfacesensitive IR spectroscopy, and molecular dynamics simulations were employed to ensure an utmost control of the Strep-MIP electrosynthesis. The functionality of this novel platform was verified with two Strep-tagged enzymes: an O2-tolerant [NiFe]-hydrogenase, and an alkaline phosphatase. The enzymes preserved their biocatalytic activities after multiple utilization confirming the efficiency of Strep-MIP as a general biocompatible platform to confine recombinant proteins for exploitation in biotechnology.
Secondary structure dependence on simulation techniques and force field parameters: from disordered to ordered proteins
(2021) Coşkuner Weber, Orkid; Çaglayan, Şule İrem
Computer simulations are used for identifying the secondary structure properties of ordered and disordered proteins. However, our recent studies showed that the chosen computer simulation protocol, simulation technique, and force feld parameter set for a disordered protein impact its predicted secondary structure properties. Here, we compare the outcome from computer simulations utilizing molecular dynamics simulations without parallel tempering techniques using various force feld parameter sets and temperature-replica exchange molecular dynamics simulations both for a model ordered and two model disordered proteins. Specifcally, the model ordered protein is the third IgG-binding domain of Protein G (GB3) and the two model disordered proteins are amyloid-?(1–40) and ?-synuclein in water. Our fndings clearly indicate that temperaturereplica exchange molecular dynamics simulations and molecular dynamics simulations without special sampling techniques yield similar results for the ordered GB3 protein whereas such agreement between simulation techniques using various force feld parameter sets could not be obtained for disordered proteins. These fndings clearly indicate that a consensus has to be reached via further development in computer simulation technique and force feld parameter sets for disordered proteins.
Identification of deleterious non-synonymous single nucleotide polymorphisms in the mRNA decay activator ZFP36L2
(2024) Akçeşme, Betül; Hekimoğlu, Hilal; Chirasani, Venkat R.; İş, Şeyma; Atmaca, Habibe Nur; Waldern, Justin M.; Ramos, Silvia B. V.
More than 4,000 single nucleotide polymorphisms (SNP) variants have been identified in the human ZFP36L2 gene, however only a few have been studied in the context of protein function. The tandem zinc finger domain of ZFP36L2, an RNA binding protein, is the functional domain that binds to its target mRNAs. This protein/RNA interaction triggers mRNA degradation, controlling gene expression. We identified 32 non-synonymous SNPs (nsSNPs) in the tandem zinc finger domain of ZFP36L2 that could have possible deleterious impacts in humans. Using different bioinformatic strategies, we prioritized five among these 32 nsSNPs, namely rs375096815, rs1183688047, rs1214015428, rs1215671792 and rs920398592 to be validated. When we experimentally tested the functionality of these protein variants using gel shift assays, all five (Y154H, R160W, R184C, G204D, and C206F) resulted in a dramatic reduction in RNA binding compared to the WT protein. To understand the mechanistic effect of these variants on the protein/RNA interaction, we employed DUET, DynaMut and PyMOL to investigate structural changes in the protein. Additionally, we conducted Molecular Docking and Molecular Dynamics Simulations to fine tune the active behaviour of this biomolecular system at an atomic level. Our results propose atomic explanations for the impact of each of these five genetic variants identified.
A tale of captopril detection based on an electrochemical mip sensör
(2024) Yarman, Aysu; Kurbanoğlu, Sevinç
Amaç: Bu çalışmada antihipertansif bir ilaç olan Kaptoprilin tespitine yönelik moleküler baskılama yöntemi ile hazırlanmış sensörler kullanılarak voltametrik bir yöntem geliştirilmesi amaçlanmıştır. Gereç ve Yöntem: Moleküler baskılama yöntemi ile camsı karbon elektroların yüzeylerinde moleküler baskınlanmış polimerler oluşturulmuş ve differansiyel puls voltammetri yöntemi ile Kaptoprilin analizi gerçekleştirilmiş, sensörün performansı incelenmiştir. Sonuç ve Tartışma: 2,62 pM teşhis sınırı değeri ile 50 pM Kaptopril seviyesine kadar doğrusal bir analiz gerçekleştirilmiştir. Seçicilik çalışmaları, Kaptoprilin, diğer girişim yapabilecek, parasetamol, askorbik asit ve L-prolin gibi maddelere göre daha yüksek elektrokimyasal cevaba sahip olduğunu göstermiştir.
Specific features of epitope-MIPs and whole-protein MIPs as illustrated for AFP and RBD of SARS-CoV-2
(2024) Yarman, Ayşe; Zhang, Xiaorong; Kovács, Norbert; Bognár, Zsófia; Gyurcsányi, Róbert E.; Bier, Frank F.; Scheller, Frieder W.
Molecularly imprinted polymer (MIP) nanofilms for alpha-fetoprotein (AFP) and the receptor binding domain (RBD) of the spike protein of SARS-CoV-2 using either a peptide (epitope-MIP) or the whole protein (protein-MIP) as the template were prepared by electropolymerization of scopoletin. Conducting atomic force microscopy revealed after template removal and electrochemical deposition of gold a larger surface density of imprinted cavities for the epitope-imprinted polymers than when using the whole protein as template. However, comparable affinities towards the respective target protein (AFP and RBD) were obtained for both types of MIPs as expressed by the KD values in the lower nanomolar range. On the other hand, while the cross reactivity of both protein-MIPs towards human serum albumin (HSA) amounts to around 50% in the saturation region, the nonspecific binding to the respective epitope-MIPs is as low as that for the non-imprinted polymer (NIP). This effect might be caused by the different sizes of the imprinted cavities. Thus, in addition to the lower costs the reduced nonspecific binding is an advantage of epitope-imprinted polymers for the recognition of proteins.
Effect of various carbon electrodes on MIP-Based sensing proteins using poly(Scopoletin): A case study of ferritin
(2024) Yarman, Ayşe
Sensitivity in the sub-nanomolar concentration region is required to determine important protein biomarkers, e.g., ferritin. As a prerequisite for high sensitivity, in this paper, the affinity of the functional monomer to the macromolecular target ferritin in solution was compared with the value for the respective molecularly imprinted polymer (MIP)-based electrodes, and the influence of various surface modifications of the electrode was investigated. The analytical performance of ferritin sensing was investigated using three different carbon electrodes (screen-printed carbon electrodes, singlewalled-carbon-nanotube-modified screen-printed carbon electrodes, and glassy carbon electrodes) covered with a scopoletin-based MIP layer. Regardless of the electrode type, the template molecule ferritin was mixed with the functional monomer scopoletin, and electropolymerization was conducted using multistep amperometry. All stages of MIP preparation were followed by evaluating the diffusional permeability of the redox marker ferricyanide/ferrocyanide through the polymer layer by differential pulse voltammetry. The best results were obtained with glassy carbon electrodes. The MIP sensor responded up to 0.5 µM linearly with a Kd of 0.30 µM. Similar results were also obtained in solution upon the interaction of scopoletin and ferritin using fluorescence spectroscopy, resulting in the quenching of the scopoletin signal, with a calculated Kd of 0.81 µM. Moreover, the binding of 1 µM ferritin led to 49.6% suppression, whereas human serum albumin caused 8.6% suppression.
Structural properties of rat intestinal fatty acid-binding protein with its dynamics: Insights into intrinsic disorder
(2024) Ballı, Öykü İrem; Cağlayan, Şule İrem; Coşkuner Weber, Orkid; Uverksy, Vladimir N.
Background: The rat intestinal fatty acid-binding protein (I-FABP) is expressed in the small intestine and is involved in the absorption and transport of dietary fatty acids. It is used as a marker for intestinal injury and is associated with various gastrointestinal disorders. I-FABP has been studied extensively using conventional experimental and computational techniques. However, the detection of intrinsically disordered regions requires the application of special sampling molecular dynamics simulations along with certain bioinformatics because conventional computational and experimental studies face challenges in identifying the features of intrinsic disorder. Methods: Replica exchange molecular dynamics simulations were conducted along with bioinformatics studies to gain deeper insights into the structural properties of I-FABP. Specifically, the C? and H? chemical shift values werecalculated, and the findings were compared to the experiments. Furthermore, secondary and tertiary structure properties were also calculated, and the protein was clustered using k-means clustering. The end-to-end distance and radius of gyration values were reported for the protein in an aqueous solution medium. In addition, its disorder tendency was studied using various bioinformatics tools. Results and Conclusion: It was reported that I-FABP is a flexible protein with regions that demonstrate intrinsic disorder characteristics. This flexibility and intrinsic disorder characteristics of IFABP may be related to its nature in ligand binding processes.
Intrinsically disordered proteins by homology modeling and replica exchange molecular dynamics simulations: A case study of amyloid-?42
(2024) Coşkuner Weber, Orkid
Homology modeling emerges as a potent tool unveiling the structural enigma of intrinsically disordered proteins (IDPs), with recent advancements such as AlphaFold2 enhancing the precision of these analyses. The process usually involves identifying homologous proteins with known structures and utilizing their templates to predict the three-dimensional architecture of the target IDP. However, IDPs lack a welldefined three-dimensional structure, and their flexibility makes it difficult to predict their conformations accurately. On the other hand, special sampling molecular dynamics simulations have been shown to be useful in defining the distinct structural properties of IDPs. Here, the structural properties of the disordered amyloid-?42 peptide were predicted using various homology modeling tools, including C-I-TASSER, ITASSER, Phyre2, SwissModel, and AlphaFold2. In parallel, extensive replica exchange molecular dynamics simulations of A?42 were conducted. Results from homology modeling were compared to our replica exchange molecular dynamics simulations and experiments to gain insights into the accuracy of homology modeling tools for IDPs used in this work. Based on our findings, none of the homology modeling tools used in this work can fully capture the structural properties of A?42. However, C-I-TASSER yields a radius of gyration and tertiary structure properties that are more in accord with the simulations and experimental data rather than I-TASSER, Phyre2, SwissModel, and AlphaFold2.
Liquid-Liquid Phase Separation Associated with Intrinsically Disordered Proteins: Experimental and Computational Tools
(2024) Uversky, Vladimir N.; Coşkuner Weber, Orkid
The phenomenon of Liquid-Liquid Phase Separation (LLPS) serves as a vital mechanism for the spatial organization of biomolecules, significantly influencing the elementary processes within the cellular milieu. Intrinsically disordered proteins, or proteins endowed with intrinsically disordered regions, are pivotal in driving this biophysical process, thereby dictating the formation of non-membranous cellular compartments. Compelling evidence has linked aberrations in LLPS to the pathogenesis of various neurodegenerative diseases, underscored by the disordered proteins’ proclivity to form pathological aggregates. This study meticulously evaluates the arsenal of contemporary experimental and computational methodologies dedicated to the examination of intrinsically disordered proteins within the context of LLPS. Through a discerning discourse on the capabilities and constraints of these investigative techniques, we unravel the intricate contributions of these ubiquitous proteins to LLPS and neurodegeneration. Moreover, we project a future trajectory for the field, contemplating on innovative research tools and their potential to elucidate the underlying mechanisms of LLPS, with the ultimate goal of fostering new therapeutic avenues for combating neurodegenerative disorders.
Structures prediction and replica exchange molecular dynamics simulations of ?-synuclein: A case study for intrinsically disordered proteins
(2024) Coşkuner Weber, Orkid
In recent years, a variety of three-dimensional structure prediction tools, including AlphaFold2, AlphaFold3, ITASSER, C-I-TASSER, Phyre2, ESMFold, and RoseTTAFold, have been employed in the investigation of intrinsically disordered proteins. However, a comprehensive validation of these tools specifically for intrinsically disordered proteins has yet to be conducted. In this study, we utilize AlphaFold2, AlphaFold3, I-TASSER, C-ITASSER, Phyre2, ESMFold, and RoseTTAFold to predict the structure of a model intrinsically disordered ?-synuclein protein. Additionally, extensive replica exchange molecular dynamics simulations of the intrinsically disordered protein are conducted. The resulting structures from both structure prediction tools and replica exchange molecular dynamics simulations are analyzed for radius of gyration, secondary and tertiary structure properties, as well as C? and H? chemical shift values. A comparison of the obtained results with experimental data reveals that replica exchange molecular dynamics simulations provide results in excellent agreement with experimental observations. However, none of the structure prediction tools utilized in this study can fully capture the structural characteristics of the model intrinsically disordered protein. This study shows that a cluster of ensembles are required for intrinsically disordered proteins. Artificial-intelligence based structure prediction tools such as AlphaFold3 and C-I-TASSER could benefit from stochastic sampling or Monte Carlo simulations for generating an ensemble of structures for intrinsically disordered proteins.
Covalent dimerization of interleukin-like epithelial-to-mesenchymal transition (EMT) inducer (ILEI) facilitates EMT, invasion, and late aspects of metastasis
(2017) Kral, Maria; Klimek, Christoph; Kutay, Betül; Timelthaler, Gerald; Lendl, Thomas; Neuditschko, Benjamin; Gerner, Christopher; Sibilia, Maria; Csiszar, Agnes
The interleukin-like epithelial-to-mesenchymal transition (EMT) inducer(ILEI)/FAM3C is a member of the highly homologous FAM3 family andis essential for EMT and metastasis formation. It is upregulated in severalcancers, and its altered subcellular localization strongly correlates withpoor survival. However, the mechanism of ILEI action, including the struc-tural requirements for ILEI activity, remains elusive. Here, we show thatILEI formed both monomers and covalent dimers in cancer cell lines andin tumors. Using mutational analysis and pulse-chase experiments, wefound that the four ILEI cysteines, conserved throughout the FAM3 fam-ily and involved in disulfide bond formation were essential for extracellularILEI accumulation in cultured cells. Modification of a fifth cysteine(C185), unique for ILEI, did not alter protein secretion, but completelyinhibited ILEI dimerization. Wild-type ILEI monomers, but not C185Amutants, could be converted into covalent dimers extracellularly uponoverexpression by intramolecular-to-intermolecular disulfide bond isomer-ization. Incubation of purified ILEI with cell culture medium showed thatdimerization was triggered by bovine serum in a dose- and time-dependentmanner. Purified ILEI dimers induced EMT and trans-well invasion ofcancer cellsin vitro. In contrast, ILEI monomers and the dimerization-defective C185A mutant affected only cell motility as detected by scratchassays and cell tracking via time-lapse microscopy. Importantly, tumorcells overexpressing wild-type ILEI caused large tumors and lung metas-tases in nude mice, while cells overexpressing the dimerization-defectiveC185A mutant behaved similar to control cells. These data show that cova-lent ILEI self-assembly is essential for EMT induction, elevated tumorgrowth, and metastasis.
Coherent organization in gene regulation a study on six networks
(2016) Sözener, Neşe Aral; Kabakçıoğlu, Alkan
Structural and dynamical fingerprints of evolutionary optimization in biological networks are still unclear. Here we analyze the dynamics of genetic regulatory networks responsible for the regulation of cell cycle and cell differentiation in three organisms or cell types each, and show that they follow a version of Hebb's rule which we have termed coherence. More precisely, we find that simultaneously expressed genes with a common target are less likely to act antagonistically at the attractors of the regulatory dynamics. We then investigate the dependence of coherence on structural parameters, such as the mean number of inputs per node and the activatory/repressory interaction ratio, as well as on dynamically determined quantities, such as the basin size and the number of expressed genes.
Intrinsically disordered proteins in various hypotheses on the pathogenesis of Alzheimer’xxs and Parkinson’xxs diseases
(Academic Press, 2019) Uversky, Vladimir N.; Coşkuner Weber, Orkid
"Dancing protein clouds: Intrinsically disordered proteins in the norm and pathology" represents a set of selected studies on a variety of research topics related to intrinsically disordered proteins. Topics in this update include structural and functional characterization of several important intrinsically disordered proteins, such as 14-3-3 proteins and their partners, as well as proteins from muscle sarcomere; representation of intrinsic disorder-related concept of protein structure-function continuum; discussion of the role of intrinsic disorder in phenotypic switching; consideration of the role of intrinsically disordered proteins in the pathogenesis of neurodegenerative diseases and cancer; discussion of the roles of intrinsic disorder in functional amyloids; demonstration of the usefulness of the analysis of translational diffusion of unfolded and intrinsically disordered proteins; consideration of various computational tools for evaluation of functions of intrinsically disordered regions; and discussion of the role of shear stress in the amyloid formation of intrinsically disordered regions in the brain.
From quantum mechanics, classical mechanics, and bioinformatics to artificial intelligence studies in neurodegenerative diseases
(Springer, 2022) Habiboğlu, Mehmet Gökhan; Coşkuner Weber, Orkid; Uversky, Vladimir N.; Teplow, David
The amyloid ?-protein is an intrinsically disordered protein that has the potential to assemble into myriad structures, including oligomers and fibrils. These structures are neurotoxic and are thought to initiate a cascade of events leading to Alzheimer’s disease. Understanding this pathogenetic process and elucidating targets for drug therapy depends on elucidation of the structural dynamics of A? assembly. In this chapter, we describe work packages required to determine the three-dimensional structures of A? and of smaller bioactive fragments thereof, which may be important in AD pathogenesis. These packages include density functional theory, Car–Parrinello molecular dynamics simulations, temperature-dependent replica exchange molecular dynamics simulations, disorder predictors based on bioinformatics, and neural network deep learning.
Chapter 33 - Methods to study the effect of solution variables on the conformational dynamics of intrinsically disordered proteins
(Elsevier Academic Press, 2022) Alıcı, Hakan; Uversky, Vladimir N.; Coşkuner Weber, Orkid; Hasekioğlu, Orkun
[Özet yok]
Current stage and future perspectives for homology modeling, molecular dynamics simulations, machine learning with molecular dynamics, and quantum computing for intrinsically disordered proteins and proteins with intrinsically disordered regions
(2024) Coşkuner Weber, Orkid; Uversky, Vladimir N.
The structural ensembles of intrinsically disordered proteins (IDPs) and proteins with intrinsically disordered regions (IDRs) cannot be easily characterized using conventional experimental techniques. Computational techniques complement experiments and provide useful insights into the structural ensembles of IDPs and proteins with IDRs. Herein, we discuss computational techniques such as homology modeling, molecular dynamics simulations, machine learning with molecular dynamics, and quantum computing that can be applied to the studies of IDPs and hybrid proteins with IDRs. We also provide useful future perspectives for computational techniques that can be applied to IDPs and hybrid proteins containing ordered domains and IDRs.
Frontotemporal dementia-related V57E mutation impairs mitochondrial function and alters the structural properties of CHCHD10
(2023) Alıcı, Hakan; Uversky, Vladimir N.; Liu, Tian; Kang, David E.; Woo, Junga Alexa; Coşkuner Weber, Orkid
The V57E pathological variant of the mitochondrial coiled-coil-helix–coiled-coil-helix domain-containing protein 10 (CHCHD10) plays a role in frontotemporal dementia. The wild-type and V57E mutant CHCHD10 proteins contain intrinsically disordered regions, and therefore, these regions hampered structural characterization of these proteins using conventional experimental tools. For the first time in the literature, we represent that the V57E mutation is pathogenic to mitochondria as it increases mitochondrial superoxide and impairs mitochondrial respiration. In addition, we represent here the structural ensemble properties of the V57E mutant CHCHD10 and describe the impacts of V57E mutation on the structural ensembles of wild-type CHCHD10 in aqueous solution. We conducted experimental and computational studies for this research. Namely, MitoSOX Red staining and Seahorse Mito Stress experiments, atomic force microscopy measurements, bioinformatics, homology modeling, and multiple-run molecular dynamics simulation computational studies were conducted. Our experiments show that the V57E mutation results in mitochondrial dysfunction, and our computational studies present that the structural ensemble properties of wild-type CHCHD10 are impacted by the frontotemporal dementia-associated V57E genetic mutation.
The impacts of the mitochondrial myopathy-associated G58R mutation on the dynamic structural properties of CHCHD10
(2023) Alıcı, Hakan; Uversky, Vladimir N.; Kang, David E.; Woo, Junga Alexa; Coşkuner Weber, Orkid
The mitochondria are responsible for producing energy within the cell, and in mitochondrial myop-athy, there is a defect in the energy production process. The CHCHD10 gene codes for a protein calledcoiled-coil-helix-coiled-coil-helix domain-containing protein 10 (CHCHD10), which is found in the mito-chondria and is involved in the regulation of mitochondrial function. G58R mutation has been shownto disrupt the normal function of CHCHD10, leading to mitochondrial dysfunction and ultimately tothe development of mitochondrial myopathy. The structures of G58R mutant CHCHD10 and howG58R mutation impacts the wild-type CHCHD10 protein at the monomeric level are unknown. Toaddress this problem, we conducted homology modeling, multiple run molecular dynamics simula-tions and bioinformatics calculations. We represent herein the structural ensemble properties of theG58R mutant CHCHD10 (CHCHD10G58R) in aqueous solution. Moreover, we describe the impacts ofG58R mutation on the structural ensembles of wild-type CHCHD10 (CHCHD10WT) in aqueous solution.The dynamics properties as well as structural properties of CHCHD10WTare impacted by the mitochon-drial myopathy-related G58R mutation. Specifically, the secondary and tertiary structure properties,root mean square fluctuations, Ramachandran diagrams and results from principal component analysisdemonstrate that the CHCHD10WTand CHCHD10G58Rproteins possess different structural ensemblecharacteristics and describe the impacts of G58R mutation on CHCHD10WT. These findings may behelpful for designing new treatments for mitochondrial myopathy.
Intrinsically disordered synthetic polymers in biomedical applications
(Polymers, 2023) Yüce-Erarslan, Elif; Kasem, Haytam; Uversky, Vladimir N.; Coşkuner Weber, Orkid; Domb, Abraham (Avi) J.
In biology and medicine, intrinsically disordered synthetic polymers bio-mimicking intrinsically disordered proteins, which lack stable three-dimensional structures, possess high structural/conformational flexibility. They are prone to self-organization and can be extremely useful in various biomedical applications. Among such applications, intrinsically disordered synthetic polymers can have potential usage in drug delivery, organ transplantation, artificial organ design, and immune compatibility. The designing of new syntheses and characterization mechanisms is currently required to provide the lacking intrinsically disordered synthetic polymers for biomedical applications bio-mimicked using intrinsically disordered proteins. Here, we present our strategies for designing intrinsically disordered synthetic polymers for biomedical applications based on bio-mimicking intrinsically disordered proteins.

Güncel Gönderiler