Biology subjects

Ciftci, H.

Publications and source records attributed to Ciftci, H..

11 recordsLinked to original sources

bioRxiv2025

Production, Purification, and Crystallization of Recombinant Epidermal Growth Factor Receptor Tyrosine Kinase Domain (EGFR-TKD) for Drug Screening Applications

Withdrawal StatementWe request the withdrawal of our preprint (MS ID#: BIORXIV/2025/657332) because the experimental data and analyses presented in the current version are not up to date. Additional experiments and methodological clarifications are required for the accurate interpretation of the results. In its current form, the manuscript may lead to misinterpretation. Therefore, to prevent the dissemination of incomplete or outdated information, we request the withdrawal of the preprint.

biochemistry↗

bioRxiv2025

Production, Purification, and Crystallization of Recombinant HER2 Tyrosine Kinase Domain (HER2-TKD) as a Platform for Structure-Based Drug Screening

The human epidermal growth factor receptor 2 tyrosine kinase domain (HER2-TKD) plays a central role in signal transduction and is a significant therapeutic target in cancer. This study aimed to produce soluble recombinant HER2-TKD in Escherichia coli to enable structural studies for drug screening applications. The HER2-TKD gene was cloned into the pET28a(+) expression vector and expressed in E. coli. Initial expression led to the formation of inclusion bodies; thus, sarcosyl was used to solubilize the aggregated protein. Several induction durations were tested to optimize soluble expression. SDS-PAGE analysis was used to monitor expression and solubilization efficiency. The recombinant protein was purified using size-exclusion chromatography and reverse affinity chromatography to remove the SUMO tag. Crystallization trials were initiated using commercial screens to obtain diffraction-quality crystals. Soluble HER2-TKD was successfully obtained after optimization of induction and solubilization conditions. Crystallization efforts are ongoing to improve crystal quality for future structural analysis. These results provide a foundation for structure-based drug discovery studies targeting HER2.

biochemistry↗

bioRxiv2025

Expression, Purification, and Crystallization of Recombinant Human ABL-1 Kinase for Structure-Based Drug Screening Applications

Abelson-1 (ABL-1) is a non-receptor tyrosine kinase that plays essential roles in various cellular processes, including proliferation, survival, differentiation and its kinase activity is tightly regulated. The dysregulated ABL-1 kinase activity is linked to disease pathogenesis like Chronic Myeloid Leukemia (CML), where the BCR::ABL-1 fusion oncoprotein drives oncogenic signaling. Due to its central role in CML pathogenesis, understanding the structure of ABL-1 is crucial for the effective management of the disease and drug development studies. This study focuses on optimizing the expression, purification and crystallization of the recombinant human ABL-1 kinase domain for its structural analysis via X-ray crystallography and structure-based drug screening applications. The human ABL-1 kinase domain, fused with a SUMO-tag, was expressed in Escherichia coli Rosetta2 BL21 using the pET28(a)+ expression vector. The ABL-1 aggregates seen under native culture conditions were successfully solubilized by mild ionic detergent sarkosyl. After obtaining soluble expression of the protein, Ni-NTA affinity chromatography was performed and high tiled of purified ABL-1 was obtained. The 6X-His-SUMO-tag of purified ABL1 was cleaved by ULP1 protease. The recombinant ABL-1 was subsequently used in crystallization trials for enlightening structural features of ABL-1 that could guide the development of novel therapeutics and drug screening platforms targeting ABL-1 in CML.

biochemistry↗

bioRxiv2025

Structural Insights into the Dynamics of Water in SOD1 Catalysis and Drug Interactions

Superoxide dismutase 1 (SOD1) is a crucial enzyme that protects cells from oxidative damage by converting superoxide radicals into H2O2 and O2. This detoxification process, essential for cellular homeostasis, relies on a precisely orchestrated catalytic mechanism involving the copper cation, while the zinc cation contributes to the structural integrity of the enzyme. This study presents the 2.3 [A] crystal structure of human SOD1 (PDB ID: 9IYK), revealing an assembly of six homodimers and twelve distinct active sites. The water molecules form a complex hydrogen-bonding network that drives proton transfer and sustains active site dynamics. Our structure also uncovers subtle conformational changes that highlight the intrinsic flexibility of SOD1, which is essential for its function. Additionally, we observe how these dynamic structural features may be linked to pathological mutations associated with amyotrophic lateral sclerosis (ALS). By advancing our understanding of hSOD1s mechanistic intricacies and the influence of water coordination, this study offers valuable insights for developing therapeutic strategies targeting ALS. Our structures unique conformations and active site interactions illuminates new facets of hSOD1 function, underscoring the critical role of structural dynamics in enzyme catalysis. Besides, we conducted molecular docking analysis using SOD1 for potential radical scavengers and Abl1 inhibitors targeting misfolded SOD1 aggregation along with oxidative stress and apoptosis, respectively. The results showed that CHEMBL1075867, a free radical scavenger derivative, showed the most promising docking results and interactions in the binding site of hSOD1, highlighting its promising role for further studies against SOD1-mediated ALS.

molecular biology↗

bioRxiv2023

4D Crystallography Captures Transient IF1-Ribosome Dynamics in Translation Initiation

Initiation factor 1 (IF1) is one of multiple key ligands involved in the initiation of mRNA translation, a highly dynamic and carefully-orchestrated process. However, details surrounding IF1 transient interactions with the small 30S ribosomal subunit remain incompletely understood despite characterization of unbound and fully-bound 30S states. Improvements in X-ray light sources and crystallographic techniques are now enabling time-resolved structural studies at near-physiological temperature and near-atomic resolution and thus the structural investigation of such dynamic processes. Here, we employed time-resolved serial femtosecond X-ray crystallography (TR-SFX) to probe the binding of IF1 to the small 30S ribosomal subunit in real time. Our time-resolved structural data demonstrates transient cryptic short-, mid-, and long-range allostery among different regions of the small 30 ribosomal subunit during IF1 binding, revealing small- and large-scale protein-target interactions and dynamics within intermediate macromolecular states at unprecedented temporal and spatial resolution. These data represent one of the first such 4D crystallographic studies assessing protein-protein and protein-RNA interactions and could serve as the basis for subsequent studies of the ribosome and of the multitudinous dynamic processes which underpin biology, and therefore, of life.

biophysics↗

bioRxiv2023

Structural Characterization of TRAF6 N-terminal for Therapeutic Uses

Tumor Necrosis Factor Receptor Associated Factors (TRAFs) are a protein family with a wide variety of roles and binding partners. Among them, TRAF6, a ubiquitin ligase, possesses unique receptor binding specificity and shows diverse functions in immune system regulation, cellular signaling, central nervous system (CNS), and tumor formation. TRAF6 consists of an N-terminal Really Interesting New Gene (RING) domain, multiple zinc fingers, and a C-terminal TRAF domain. RING domain and zinc fingers mediate the activation of nuclear factor kappa B (NF-{kappa}B), which has essential roles in the regulation of inflammatory responses, proliferation, differentiation, migration, cell adhesion, and apoptosis. Therefore, it has been found that TRAF6 is overexpressed in various types of cancer including pancreatic, liver, lung, head and neck, breast, colorectal cancers, and melanoma along with inflammatory, autoimmune and neurodegenerative disorders. Furthermore, TRAF6 is an important therapeutic target for numerous disorders and structural studies of this protein are crucial for the development of next-generation therapeutics. Here, we present a TRAF6 N-terminal structure determined at the Turkish Light Source "Turkish DeLight" to 2.6 [A] resolution at cryogenic temperature. This structure offers insight into the domain organization and zinc-binding, which are critical for protein function. Since the RING domain and the zinc fingers are key targets for TRAF6 therapeutics, structural insights are crucial for future research.

molecular biology↗

bioRxiv2022

Comparative Study of High-resolution LysB29(Nε-myristoyl) des(B30) Insulin Structures Display Novel Dynamic Causal Interrelations in Monomeric-Dimeric Motions

The treatment of insulin-dependent diabetes mellitus is characterized by artificial supplementation of pancreatic {beta}-cell ability to regulate sugar levels in the blood. Even though various insulin analogs are crucial for reasonable glycemic control, understanding the dynamic mechanism of the insulin analogs may help to improve the best-protracted insulin analog to assist people with Type 1 Diabetes (T1D) to live comfortably while maintaining tight glycemic control. Here we present the high-resolution crystal structure of NN304, known as insulin detemir, to 1.7 [A] resolution at cryogenic temperature. We computationally further investigated our crystal structures monomeric-dimeric conformation and dynamic profile by comparing it with a previously available detemir structure (PDB ID: 1XDA). Our structure (PDB ID: 8HGZ) obtained at elevated pH provides electrostatically triggered minor movements in the equilibrium between alternate conformational substates compared to the previous structure, suggesting it might induce an intermediate state in the dissociation pathway of the insulin detemirs hexamer:dihexamer equilibrium. Supplemented with orientational cross-correlation analysis by Gaussian Network Model (GNM), this alternate oligomeric conformation offers the distinct cooperative motions originated by loose coupling of distant conformational substates of a protracted insulin analog that has not been previously observed.

biophysics↗

bioRxiv2022

Rapid and High Resolution Ambient Temperature Structure Determination at Turkish Light Source

High-resolution biomacromolecular structure determination is essential to better understand protein function and dynamics. Serial crystallography is an emerging structural biology technique which has fundamental limitations due to either sample volume requirements or immediate access to the competitive X-ray beamtime. Obtaining a high volume of well-diffracting, sufficient-size crystals while mitigating radiation damage remains a critical bottleneck of serial crystallography. As an alternative, we introduce the plate-reader module adapted for using a 72-well Terasaki plate for biomacromolecule structure determination at a convenience of a home X-ray source. We also present the first ambient temperature lysozyme structure determined at the Turkish Light Source (Turkish DeLight). The complete dataset was collected in 18.5 mins with resolution extending to 2.39 [A] and 100% completeness. Combined with our previous cryogenic structure (PDB ID: 7Y6A), the ambient temperature structure provides invaluable information about the structural dynamics of the lysozyme. Turkish DeLight provides robust and rapid ambient temperature biomacromolecular structure determination with limited radiation damage.

biophysics↗

bioRxiv2022

Cryogenic X-ray crystallographic studies of biomacromolecules at Turkish Light Source: Turkish DeLight

X-ray crystallography is a robust and powerful structural biology technique that provides high-resolution atomic structures of biomacromolecules. Scientists use this technique to unravel mechanistic and structural details of biological macromolecules (e.g. proteins, nucleic acids, protein complexes, protein-nucleic acid complexes, or large biological compartments). Since its inception, single-crystal cryo-crystallography has never been performed in Turkiye due to the lack of a single-crystal X-ray diffractometer. The X-ray diffraction facility recently established at the University of Health Sciences, Istanbul, Turkiye will enable Turkish and international researchers to easily perform high-resolution structural analysis of biomacromolecules from single crystals. Here, we describe the technical and practical outlook of a state-of-the-art home-source X-ray, using lysozyme as a model protein. The methods and practice described in this article can be applied to any biological sample for structural studies. Therefore, this article will be a valuable practical guide from sample preparation to data analysis.

biophysics↗

bioRxiv2021

Structural insight into host plasma membrane association and assembly of HIV-1 Matrix protein

HIV-1 continues to be a global health concern since AIDS was first recognized by the World Health Organization (WHO). It is estimated that there were 38 million people infected with HIV-1 and 1.5 million deaths in 2019 alone. A better understanding of the details of the HIV late-stage life cycle, involving Pr55Gag attachment to the membrane for the further oligomerization to release virion, will provide us new avenues for potential treatment. Inositol hexakisphosphate (IP6) is an abundant endogenous cyclitol molecule and its binding was linked to the oligomerization of Pr55Gag via the MA domain. However, the binding site of IP6 on MA was unknown and the structural details of this interaction were missing. Here, we present three high-resolution crystal structures of the MA domain in complex with IP6 molecules to reveal its binding mode. Additionally, extensive Differential Scanning Fluorimetry analysis combined with cryo- and ambient-temperature X-ray crystallography and computational biology identify the key residues that participate in IP6 binding. Our data provide novel insights about the multilayered HIV-1 virion assembly process that involves the interplay of IP6 with PIP2, a phosphoinositide essential for the membrane binding of Pr55Gag. IP6 and PIP2 have neighboring alternate binding sites within the same highly basic region (residues 18-33). This indicates that IP6 and PIP2 bindings are not mutually exclusive and may play a key role in coordinating virion particles membrane localization. Based on our three different IP6-MA complex crystal structures, we propose a new model that involves the IP6 coordination of the oligomerization of outer MA and inner CA domain 2D layers during assembly and budding.

microbiology↗

bioRxiv2018

Aminoglycoside ribosome interactions reveal novel conformational states at ambient temperature

The bacterial 30S ribosomal subunit is a primary antibiotic target. Despite decades of discovery, the mechanisms by which antibiotic binding induces ribosomal dysfunction are not fully understood. Ambient temperature crystallographic techniques allow more biologically relevant investigation of how local antibiotic binding site interactions trigger global subunit rearrangements that perturb protein synthesis. Here, the structural effects of 2-deoxystreptamine (paromomycin and sisomicin), a novel sisomicin derivative, N1-methyl sulfonyl sisomicin (N1MS) and the non-deoxystreptamine (streptomycin) aminoglycosides on the ribosome at ambient and cryogenic temperatures were examined. Comparative studies led to three main observations. First, individual aminoglycoside-ribosome interactions in the decoding center were similar for cryogenic vs ambient temperature structures. Second, analysis of a highly conserved GGAA tetraloop of h45 revealed aminoglycoside-specific conformational changes, which are affected by temperature only for N1MS. We report the h44/h45 interface in varying states, that is, engaged, disengaged and in equilibrium. Thirdly, we observe aminoglycoside-induced effects on 30S domain closure, including a novel intermediary closure state, which is also sensitive to temperature. Analysis of three ambient and five cryogenic crystallography datasets reveal a correlation between h44/h45 engagement and domain closure. These observations illustrate the role of ambient temperature crystallography in identifying dynamic mechanisms of ribosomal dysfunction induced by local drug-binding site interactions. Together these data identify tertiary ribosomal structural changes induced by aminoglycoside binding that provides functional insight and targets for drug design.

biophysics↗