Biology subjects

Gocenler, O.

Publications and source records attributed to Gocenler, O..

7 recordsLinked to original sources

bioRxiv2025

M9 Medium Composition Alters E. coli Metabolism During Recombinant Expression

M9 minimal media and its enhanced variants (M9+ and M9++) are widely used for recombinant protein expression in Escherichia coli, particularly for isotopic labeling required in structural biology techniques such as NMR spectroscopy. This study investigates how different compositions of M9-based media (M9, M9+, and M9++) influence bacterial growth, metabolic stress, and central carbon metabolism during recombinant expression of the protein. Using 1D 1H NMR spectroscopy and multivariate statistical analysis, we observed distinct media-dependent metabolic shifts. Standard M9 exhibited limited bacterial growth and heightened stress-related fermentation, indicated by high ethanol and acetate levels. In contrast, M9+ significantly increased biomass but promoted pronounced overflow metabolism. M9++ presented intermediate biomass levels and markedly reduced overflow metabolites, favoring biosynthesis pathways, notably increasing valine, acetoin, and formate concentrations. These findings suggest that further optimization of glucose concentration, nitrogen sources, and phosphate buffering could significantly improve the metabolic balance of M9++, creating an enhanced medium tailored for efficient, high-quality recombinant protein expression and isotopic labeling in E. coli.

biochemistry↗

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↗

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↗

bioRxiv2021

Case Study of High-Throughput Drug Screening and Remote Data Collection for SARS-CoV-2 Main Protease by Using Serial Femtosecond X-ray Crystallography

Since early 2020, COVID-19 has grown to affect the lives of billions globally. A worldwide investigation has been ongoing for characterizing the virus and also for finding an effective drug and developing vaccines. As time has been of the essence, a crucial part of this research has been drug repurposing; therefore, confirmation of in-silico drug screening studies has been carried out for this purpose. Here we demonstrated the possibility of screening a variety of drugs efficiently by leveraging a high data collection rate of 120 images/second with the new low-noise, high dynamic range ePix10k2M Pixel Array Detector installed at the Macromolecular Femtosecond Crystallography (MFX) instrument at the Linac Coherent Light Source (LCLS). The X-ray Free-Electron Laser (XFEL) is used for remote high-throughput data collection for drug repurposing of the main protease (Mpro) of SARS-CoV-2 at ambient temperature with mitigated X-ray radiation damage. We obtained multiple structures soaked with 9 drug candidate molecules in two crystal forms. Although our drug binding attempts failed, we successfully established a high-throughput Serial Femtosecond X-ray crystallographic (SFX) data collection protocol.

molecular biology↗

bioRxiv2020

Near-Physiological-Temperature Serial Femtosecond X-ray Crystallography Reveals Novel Conformations of SARS-CoV-2 Main Protease Active Site for Improved Drug Repurposing

The COVID19 pandemic has resulted in 25+ million reported infections and nearly 850.000 deaths. Research to identify effective therapies for COVID19 includes: i) designing a vaccine as future protection; ii) structure-based drug design; and iii) identifying existing drugs to repurpose them as effective and immediate treatments. To assist in drug repurposing and design, we determined two apo structures of Severe Acute Respiratory Syndrome CoronaVirus-2 main protease at ambienttemperature by Serial Femtosecond X-ray crystallography. We employed detailed molecular simulations of selected known main protease inhibitors with the structures and compared binding modes and energies. The combined structural biology and molecular modeling studies not only reveal the dynamics of small molecules targeting main protease but will also provide invaluable opportunities for drug repurposing and structure-based drug design studies against SARS-CoV-2. One Sentence SummaryRadiation-damage-free high-resolution SARS-CoV-2 main protease SFX structures obtained at near-physiological-temperature offer invaluable information for immediate drug-repurposing studies for the treatment of COVID19.

biophysics↗