Biology subjects

Kahraman, K.

Publications and source records attributed to Kahraman, K..

7 recordsLinked to original sources

bioRxiv2025

Intrinsic conformational equilibria position arrestin-2 for activation

Arrestins regulate G protein-coupled receptor (GPCR) signaling by undergoing large-scale conformational rearrangements, yet the solution-state equilibria that underlie arrestin pre-activation remain poorly defined. Here, we use methyl-specific nuclear magnetic resonance spectroscopy, temperature-dependent chemical shift analysis, and relaxation measurements to characterize the intrinsic conformational landscape of full-length human arrestin-2 in solution. We identify two distinct equilibria with separable thermodynamic and kinetic signatures. A slow, enthalpically-favored process sensed by interdomain isoleucines I241 and I317 populates an active-like, interdomain-twisted conformation at physiological temperatures. In parallel, a faster, globally distributed equilibrium consistent with C-terminal tail release exhibits opposing thermodynamic behavior. Dynamic analyses reveal localized rigidification in the active-like minor states despite arrestins overall flexibility, while backbone relaxation data indicate widespread s-ms conformational exchange. Together, these results demonstrate that arrestin-2 intrinsically samples activation-relevant conformations in the absence of binding partners, providing a solution-state framework for arrestin pre-activation and signaling competence.

biophysics↗

bioRxiv2025

Role of Uba7-UFD in E1-E2 ISGylation: NMR-Based Insights into E2 Binding Dynamics

The ISGylation pathway, a key post-translational modification, plays a pivotal role in the innate immune response by covalently attaching ISG15 to target proteins. This cascade involves a series of enzymatic steps, including activation by the E1 enzyme Uba7, conjugation by the E2 enzyme UbcH8, and ligation by an E3 ligase. Central to this process is the ubiquitin-fold domain (UFD) of Uba7, which facilitates the transfer of ISG15 to UbcH8. However, the structural and mechanistic details of this interaction remain poorly understood. In this work, we present the solution NMR structure and functional analysis of the ubiquitin-fold domain of human Uba7, the E1 enzyme in the ISGylation cascade. Through detailed NMR titration experiments and mutational studies, we mapped the interaction surface of Uba7-UFD with UbcH8, identifying key residues and their contributions to the E1-E2 interaction. We found that Uba7-UFD is flexible in its free form; this flexibility is conserved across ubiquitin-like systems but shows a regional shift that may play an important role in correct E2 and Ubl selection. Chemical shift perturbation and mutational analysis further demonstrate the importance of specific residues, particularly Cys996, in maintaining UFDs structural integrity and binding capacity. Additionally, mutations designed to alter the flexibility and length of the loop region between UFD and UbcH8 show significant effects on binding, indicating that these regions are crucial for efficient E2 recruitment. These findings provide new insights into the mechanistic basis of E2 enzyme selection in ISGylation and underscore the functional relevance of dynamic structural transitions in E1-E2 complex formation. Statement of SignificanceISGylation is a key ubiquitin-like modification pathway essential for antiviral defense, immune regulation, and protein quality control. However, the molecular principles that govern communication between the E1 and E2 enzymes in this pathway remain poorly defined. Here, we present the first NMR structure of the human Uba7 ubiquitin-fold domain (UFD) and its interaction with UbcH8. Our findings reveal how structural flexibility within the UFD is crucial for specific and efficient E2 recognition, providing fundamental insight into the dynamic mechanism of ISGylation and advancing our understanding of ubiquitin-like enzyme cascades relevant to cellular regulation and disease.

biochemistry↗

bioRxiv2024

Exploring the Biochemical Landscape of Bacterial Medium with Pyruvate as the Exclusive Carbon Source for NMR Studies

The use of Escherichia coli for recombinant protein production is a cornerstone in structural biology, particularly for nuclear magnetic resonance (NMR) spectroscopy studies. Understanding the metabolic behavior of E. coli under different carbon sources is critical for optimizing isotope labeling strategies, which are essential for protein structure determination by NMR. Recent advancements, such as mixed pyruvate labeling, have enabled improved backbone resonance assignment in large proteins, making selective isotopic labeling strategies more important than ever for NMR studies. In this study, we aimed to investigate the metabolic adaptations of E. coli when grown on pyruvate as the sole carbon source, a common condition used to achieve selective labeling for NMR spectroscopy. Using NMR-based metabolomics, we tracked key metabolic shifts throughout the culture process to better understand how pyruvate metabolism affects protein production and isotopic labeling. Our results reveal that pyruvate is rapidly depleted before IPTG induction, while acetate and lactate accumulate due to overflow metabolism. These byproducts persist after induction, indicating that pyruvate is diverted into waste pathways, which limits its efficient use in isotope incorporation. This metabolic inefficiency presents a challenge for isotopic labeling protocols that rely on pyruvate as a carbon source for NMR studies. Our results highlight the need to fine-tune pyruvate supplementation to improve metabolic efficiency and isotopic labeling, making this study directly relevant to optimizing protocols for NMR studies involving protein structure determination. These insights provide valuable guidance for enhancing the quality and yield of isotopically labeled proteins in NMR spectroscopy.

biochemistry↗

bioRxiv2023

Characterization of Turkish Pine Honey and Differentiation from other floral honey types from Oceania, Aegean, West Coast by NMR spectroscopy and Chemometric Analysis

Honey is a viscous, supersaturated sugar solution produced by bees through the enzymatic transformation of nectar from flowers, containing a complex mixture of carbohydrates, organic acids, enzymes, and other minor constituents. Although honey has been used for thousands of years for its nutritional and medicinal properties, it has been the subject of increasing attention in recent years due to increasing adulterated honey production. Consequently, assessment of honey quality and authenticity has become essential to ensure consumer confidence of regional honey and to perverse the practice of authentic honey production. In this study, we employed nuclear magnetic resonance (NMR) spectroscopy and chemometric analysis to characterize Turkish Pine honey and compare it to flower honey originating from the Oceania, Aegean, and West Coast of North America regions. Utilizing 1H NMR spectroscopy, the chemical profile of Turkish Pine honey was characterized, and unique peaks were found. Additionally, PLS-DA statistical analysis was employed to further investigate the distinction of Turkish Pine honey among other floral and regional honeys. Upon completion of our statistical analysis, we were able to effectively distinguish Turkish Pine honey from other regional honey types, allowing us to formulate a universal test for authenticity of Turkish Pine Honey.

biochemistry↗

bioRxiv2023

Characterizing the monomer-dimer equilibrium of UbcH8/Ube2L6: A combined SAXS and NMR study

Interferon-stimulated gene-15 (ISG15) is an interferon-induced protein with two ubiquitin-like (Ubl) domains linked by a short peptide chain, and the conjugated protein of the ISGylation system. Similar to ubiquitin and other Ubls, ISG15 is ligated to its target proteins through a series of E1, E2, and E3 enzymes known as Uba7, Ube2L6/UbcH8, and HERC5, respectively. Ube2L6/UbcH8 plays a literal central role in ISGylation, underscoring it as an important drug target for boosting innate antiviral immunity. Depending on the type of conjugated protein and the ultimate target protein, E2 enzymes have been shown to function as monomers, dimers, or both. UbcH8 has been crystalized in both monomeric and dimeric forms, but the functional state is unclear. Here, we used a combined approach of small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy to characterize UbcH8s oligomeric state in solution. SAXS revealed a dimeric UbcH8 structure that could be dissociated when fused N-terminally to glutathione S-transferase. NMR spectroscopy validated the presence of a concentration-dependent monomer-dimer equilibrium and suggested a backside dimerization interface. Chemical shift perturbation and peak intensity analysis further suggest dimer-induced conformational dynamics at E1 and E3 interfaces - providing hypotheses for the proteins functional mechanisms. Our study highlights the power of combining NMR and SAXS techniques in providing structural information about proteins in solution.

biochemistry↗

bioRxiv2022

Biomolecular solution X-ray scattering at n2STAR Beamline

Small angle X-ray Scattering (SAXS) is a method for determining basic structural characteristics such as size, shape, and surface of particles. SAXS can generate low resolution models of biomolecules faster than any other conventional structural biology tools. SAXS data is mostly collected in synchrotron facilities to obtain the best scattering data possible however home source SAXS devices can also generate valuable data when optimized properly. Here, we examined sample data collection and optimization at home source SAXS beamline in terms of concentration, purity, and the duration of data acquisition. We validated that high concentration, monodisperse and ultra pure protein samples obtained by size exclusion chromatography are necessary for generating viable SAXS data using home source beamline. Longer data collection time does not always generate higher resolutions but at least one hour is required for generating a feasible model from SAXS data. Furthermore, with small optimizations both during data collection and later data analysis SAXS can determine properties such as oligomerization, molecular mass, and overall shape of particles in solution under physiological conditions.

biophysics↗

bioRxiv2019

A chromosome-scale genome assembly of European Hazel (Corylus avellana L.) reveals targets for crop improvement

European hazelnut (Corylus avellana L.) is a tree crop of economic importance worldwide, but especially to northern Turkey, where the majority of production takes place. Hazelnut production is currently challenged by environmental stresses such as a recent outbreak of severe powdery mildew disease; furthermore, allergy to hazelnuts is an increasing health concern in some regions.\n\nIn order to provide a foundation for utilizing the available hazelnut genetic resources for crop improvement, we produced the first fully assembled genome sequence and annotation for a hazelnut species, from Corylus avellana cv. Tombul, one of the most important Turkish varieties. A hybrid sequencing strategy combining short reads, long reads and proximity ligation methods enabled us to resolve heterozygous regions and produce a high-quality 370 Mb assembly that agrees closely with cytogenetic studies and genetic maps of the 11 C. avellana chromosomes, and covers 97.8% of the estimated genome size. The genome includes 28,409 high-confidence protein-coding genes, over 20,000 of which were functionally annotated based on homology to known plant proteins. We focused particularly on gene families encoding hazelnut allergens, and the MLO proteins that are an important susceptibility factor for powdery mildew. The complete assembly enabled us to differentiate between members of these families and identify novel homologs that may be important in mildew disease and hazelnut allergy. These findings provide examples of how the genome can be used to guide research and develop effective strategies for crop improvement in C. avellana.

genomics↗