Biology subjects

Dag, C.

Publications and source records attributed to Dag, C..

11 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

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↗

bioRxiv2025

Structural Insights into the Trimeric Assembly of the HERC5 HECT Domain through SAXS Analysis

HERC5 is an interferon-induced E3 ubiquitin ligase that mediates ISGylation, a critical post-translational modification involved in regulating antiviral immune responses. Its HECT (Homologous to the E6-AP Carboxyl Terminus) domain is essential for substrate recognition and the catalytic transfer of ubiquitin or ubiquitin-like proteins. Members of the HERC protein family are structurally defined by a C-terminal HECT domain and N-terminal RCC1-like (Regulator of Chromosome Condensation 1) domains. Despite its biological importance, the oligomerization behavior of the HERC5 HECT domain remains poorly understood. In this study, we examined the oligomeric state of the HERC5 HECT domain in solution using affinity and size-exclusion chromatography, followed by small-angle X-ray scattering (SAXS) at a protein concentration of 2.0 mg/ml and temperature of 10{degrees}C. SAXS data analysis revealed that the HERC5 HECT domain predominantly adopts a trimeric assembly. This conclusion is supported by Guinier and Kratky analyses, distance distribution functions, and ab initio shape reconstructions. These findings suggest that trimerization may play a regulatory role in the functional activity of HERC5, contributing to a deeper understanding of the structural mechanisms governing HECT-type E3 ligases.

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↗

bioRxiv2021

Cooperative Allostery and Structural Dynamics of Streptavidin at Cryogenic- and Ambient-temperature

Multimeric protein assemblies are abundant in nature. Streptavidin is an attractive protein that provides a paradigm system to investigate the intra- and intermolecular interactions of multimeric protein complexes. Also, it offers a versatile tool for biotechnological applications. Here, we present two apo-streptavidin structures, the first one is an ambient temperature Serial Femtosecond X-ray crystal (Apo-SFX) structure at 1.7 [A] resolution and the second one is a cryogenic crystal structure (Apo-Cryo) at 1.1 [A] resolution. These structures are mostly in agreement with previous structural data. Combined with computational analysis, these structures provide invaluable information about structural dynamics of apo streptavidin. Collectively, these data further reveal a novel cooperative allostery of streptavidin which binds to substrate via water molecules that provide a polar interaction network and mimics the substrate biotin which displays one of the strongest affinities found in nature.

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↗

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↗