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Bu çalışmada, 2014-2016 yılları arasında Türkiye'nin 5 bölgesinde yer alan 11 ilinden toplamda 264 farklı zeytin örneği toplanmıştır. Toplanan zeytin örneklerinin tümü aynı şartlar altında işlenerek monovaryete natürel sızma zeytinyağı örnekleri elde edilmiştir. Ayrıca 45 adet ticari monovaryete natürel sızma zeytinyağı örneklenmiş ve toplamda 309 monovaryete natürel sızma zeytinyağı örneği kullanılarak NMR tabanlı otantisite ve orijin belirleme çalışmaları gerçekleştirilmiştir.Çalışmanın ilk aşamasında, Türkiye'nin çeşitli bölgelerinden toplanan zeytin örneklerinden elde edilen monovaryete zeytinyağlarının NMR spektroskopisi ile analizleri gerçekleştirilerek Türkiye menşeli monovaryete natürel sızma zeytinyağları için NMR verilerine dayanan bir veri bankası oluşturulmuştur. Oluşturulan NMR veri tabanı kullanılarak Edremit Yağlık, Memecik, Gemlik ve Nizip Yağlık zeytin çeşitlerinin OPLS-DA modellemeleri ile otantisite ve coğrafi işaretlemeleri gerçekleştirilmiştir. Bunun yanında, veri tabanının ticari örnekler ile uyumluluğu da OPLS-DA modellemeleri ile test edilmiştir. Çalışmanın ikinci kısmında ise; toplanan zeytinlerden elde edilen ve ticari olarak temin edilen zeytinyağı örneklerinin bazılarının sterol, tokoferol ve yağ asidi analizleri gerçekleştirilerek biyokimyasal profilleri çıkartılmış ve biyokimyasal analiz verilerinin OPLS-DA modellerinin otantisite belirlemede kullanılabilirliği analiz edilmiştir. Ayrıca, NMR spektroskopisi verilerinin OPLS-DA modellemeleri ile biyokimyasal analiz verilerinin OPLS-DA modellemeleri karşılaştırılarak, hangi yöntemin otantisite belirlemede daha etkin olduğu test edilmiştir. Yapılan çalışmalar sonucunda, 1D 1H NMR verileri kullanılarak Edremit Yağlık, Nizip Yağlık ve Memecik zeytin çeşitlerinden elde edilen monovaryete natürel sızma zeytinyağları OPLS-DA yöntemi kullanılarak başarı ile sınıflandırılmıştır. İlaveten, oluşturulan veri tabanı kullanılarak ticari örneklerin otantisitelerinin belirlenmesi çalışmaları da başarılı sonuç vermiştir. Sonuç olarak bu çalışma, hem NMR spektroskopisi verilerinin hem de biyokimyasal analiz verilerinin, Türkiye meşeli zeytinyağlarının otantisite ve coğrafi işaretleme çalışmalarında kullanım için uygun olduğunu göstermiştir. Ancak, NMR spektroskopisi verileri kullanılarak elde edilen OPLS-DA modellemelerinin, yağ asidi veya sterol kompozisyon profilleri kullanılarak elde edilen OPLS-DA modellemelerine göre zeytinleri çeşitlerine göre gruplandırmada daha başarılı olduğunu göstermektedir. In this study, a total of 264 different olive samples were collected from 11 provinces in 5 regions of Turkey between the years of 2014 and 2016. All of the olive samples were processed under the same conditions to obtain monocultivar natural extra virgin olive oil samples. Additionally, 45 commercial monocultivar virgin olive oil were also collected. NMR based authenticity and origin determination studies were carried out with a total of 309 monocultivar natural extra virgin olive oil samples. In the first part of the study, monocultivar olive oil samples obtained from olive samples collected from various regions of Turkey were analyzed by NMR spectroscopy to build an NMR based databank of monocultivar natural virgin olive oils of Turkey. Using this NMR database, OPLS-DA modelling was used to determine authenticity and geographical origins of the Edremit Yağlık, Memecik, Gemlik and Nizip Yağlık olive varieties. Moreover, the compatibility of the database with commercial olive oil samples was also tested using OPLS-DA modellings. In the second part of the study, sterol, tocopherol and fatty acid analysis of selected oil samples obtained from collected olives and supplied as commercial olive oils were carried out to extract their biochemical profiles. The ability of OPLS-DA modeling of the biochemical analysis data in grouping olive oils according to olive variety was also tested. In addition, OPLS-DA modelings obtained from NMR data and biochemical analysis data were compared to determine which method is more effective in authenticity analysis. As a result of these studies, monocultivar natural extra virgin olive oils obtained from Edremit Yağlık, Nizip Yağlık and Memecik olive varieties were successfully classified by OPLS-DA method using the 1D 1H NMR data. In addition, the database has also been successfully used in determining the authenticity of commercial samples.As a result, this study demonstrated that both NMR spectroscopy data and biochemical analysis data can be used in authenticity analysis and geographical marking studies of Turkish olive oils. Moreover, OPLS-DA models obtained from NMR spectroscopy data is more successful in grouping olive oils according to the olive variety than OPLS-DA models obtained from fatty acid or sterol composition data. 187

Ubiquitin (Ub) ve Ub benzeri (Ubl) proteinler bir dizi enzimatik reaksiyonlar ile ökaryotik proteinleri post translasyonel modifikasyonlara uğratan proteinlerdir. Hedef proteine Ub/Ubl konjugasyonu E1 aktive edici, E2 konjuge edici ve E3 ligazlar olarak adlandırılan üç enzim sınıfının aktivitesini gerektirir ve her bir Ub/Ubl protein için spesifik E1, E2 ve E3 enzimleri mevcuttur. Ub ve Ubl proteinler bağlandığı hedef proteinleri farklı yol ve işlevlere yönlendirirler. Hedef proteinin hangi yol ve işleve yönlendirileceği ise hedef proteine Ub/Ubl proteinin hangi aminoasitten bağlandığına ve bağlanan Ub/Ubl moleküllerinin sayısına göre çeşitlilik göstermektedir.İnterferonca uyarılan gen 15 (ISG15) bağışıklık sisteminde rol oynayan ubiquitin benzeri bir proteindir. ISG15'in ökaryotik proteinlere kovalent olarak bağlanması ile gerçekleşen post translasyonel modifikasyon ISGlasyon olarak adlandırılır. İnsan ISG15'i için spesifik E1 enzimi Ube1L'dir. Ub/Ubl konjugasyon sistemlerinde E1 enzimleri yapısal ve işlevsel bakımdan benzerlik göstermektedir. E1 aktive edici enzimler genellikle üç domainden oluşurlar; adenilasyon domaini, katalitik sistein domaini ve karboksil terminal ubiquitin benzeri beta grasp/ubiquitin fold (UF) domaini. Literatürde insan Ube1L enzimi ile ilgili yapısal bir çalışma mevcut değildir. Ancak diğer Ub/Ubl protinler için spesifik E1 enzimleri ile yapılan çalışmalarda, UF domaininin E2 ile etkileşimde ve doğru E2 enziminin seçilmesinde gerekli olduğu gösterilmiştir. Sistemler arasında seçiciliğin nasıl sağlandığının anlaşılmasında yapısal çalışmaların rolü büyüktür ve Ube1L enzimi ile ilgili yapısal çalışmalar bu seçiciliğin anlaşılmasına yardımcı olacaktır.Bu tezin ilk bölümünde, ISGylasyonda aktive edici E1 enzimi olarak işlev yapan Ube1L'nin ubiquitin benzeri domaininin (UFD) 3 boyutlu NMR solüsyon yapısının belirlenmesine yönelik çalışmalar gerçekleştirilmiştir. Ube1L-UFD için gerekli NMR verileri kaydedilmiş, rezonans atamaları, verilerin analizi ve yapı hesaplamaları kısmi olarak tamamlanmıştır. Bu çalışmalar sonucunda, Ube1L-UFD'nin, diğer ubiquitin benzeri proteinlerde olduğu gibi beta-grasp fold olarak adlandırılan yapıya katlandığı ortaya koyulmuştur.Tezin ikinci bölümünde Ub/Ubl sisteminin hedeflerinden birisi olan insan RIG-I (retinoic acid inducible gene-1) proteininin modifikasyona uğrayan CAR (caspase activation and recruitment) domainleri ve bu modifikasyonda işlev yapan E3 enzimlerinin (Trim25 ve RNF125) yapısal çalışmalarda kullanılmak amacıyla üretilmesine yönelikçalışmalar gerçekleştirilmiştir. RIG-1, RNA virüsleri tarafından enfekte olmuş hücrelerde, viral RNA moleküllerinin tanınmasında ve savunma mekanizmasının harekete geçirilmesinde birinci basamakta yer alır. RIG-I'in regülasyonunda ubiquitilasyon önemli bir rol oynar. RIG-1'in ubiquitilasyonunda görev aldığı bilinen iki farklı E3 enzimi mevcuttur; Trim25 ve RNF125. Trim25 ve RNF125 aracılıklı ubiqutilasyonun RIG-I'i farklı sinyal yollarına yönlendirdiği bilinmektedir. RNF125 aracılıklı ubiquitilasyon RIG-I'i degredasyon için proteozoma yönlendirirken, Trim25 aracılıklı ubiquitilasyon RIG-1 bağımlı viral bağışıklığın harekete geçirilmesinde gereklidir.Bu bölümünde, RIG-1 CAR domainlerini ve Trim25-SPRY domainini ve RNF125'i kodlayan genler E. Coli ekspresyon vektörleri içerisine klonlanmış, bakteriyel sistemde ekspresyonları ve kromatografik yöntemlerle saflaştırılmaları gerçekleştirilmiştir. Ayrıca RIG-I CARDs 15N-işaretli olarak üretilmiş ve NMR yapısal çalışmalar için uygun olup olmadığı test edilmiştir. Bu çalışmalar sonucunda, Trim25-SPRY ve RNF125 proteinlerinin çözünürlüklerinin düşük olduğu, denenen solüsyon koşullarında kararsız oldukları ve hızla çöktükleri gözlenmiştir. RIG-I CARDs'ın ise kaydedilen 15N-1H HSQC spektrumundan, test edilen NMR koşullarında kararlı bir üç boyutlu yapıya sahip olmadığı belirlenmiştir. Ubiquitin (Ub) and ubiquitin-like (Ubl) proteins are common post-translational modifiers of eukaryotic proteins. Ub/Ubl conjugation to target proteins occurs via similar enzymatic cascades and requires the activity of three enzyme classes, namely E1 activating enzymes, E2 conjugating enzymes and E3 ligases. Each Ub/Ubl protein has its own specific set of E1, E2 and E3 enzymes and each Ub/Ubl protein directs its target to different pathways and functions. Which pathway or function target is directed to depends both on the attachment position of Ub/Ubl on the target and the number of attached Ub/Ubl molecules.Interferon stimulated gene 15 is a ubiquitin-like protein involved in immune defense. Post-translational modification of eukaryotic proteins by covalent attachment of ISG15 is called ISGlation. Specific E1 enzyme for human ISG15 is Ube1L. E1 enzymes in Ub/Ubl conjugation systems display similar structural and functional aspects. They generallyconsist of three different domains: adenilation domain, the catalytic cysteine domain and the carboxyl terminal ubiquitin-like beta-grasp (ßG) / ubiquitin fold (UF). There is no structural work in the literature for human Ube1L enzyme yet. However, structural studies on other Ub/Ubl specific E1s show that UF domain is important for interacting E2s and necessary in selecting the correct E2 for the pathway. Structural studies play important roles in understanding how specificity is achieved in various conjugation systems. Structural studies on human Ube1L will help to understand this specificity.In the second part of this thesis, production of human RIG-1 CAR (caspase activation and recruitiment) domains, which are the targets of various Ub/Ubl modification systems, and two E3 ligases (Trim25 and RNF125), which are involded in ubiquitylation of RIG-1 CARDs (CAR domains), were carried out to use in structural studies. In cells infected with RNA viruses, RIG-1 is involved in the first step in recognition of viral RNA molecules and activation of host defense. Ubiquitylation plays an important role in RIG-1 regulation. There are two different E3 enzymes that are known to function in RIG-1 ubiquitylation; Trim25 and RNF125. Ubiquitylation of RIG-1 CAR domains by different E3s, targets RIG-1 to different signal pathways. While ubiquitylation by RNF125 targets RIG-1 to proteosome for degredation, ubiquitylation by Trim25 is necessary for activation of RIG-1 dependent viral defense system.In this part, genes coding for RIG-1 CARDs, Trim25-SPRY domain and RNF15 were cloned into E. Coli expression vectors, bacterial protein expressions and purification of target proteins by chromatographic methods were carried out. In addition, 15N-labeled RIG-I CARDs was produced to test whether free domains were suitable for structural studies. Result of these studies showed that both Trim25-SPRY and RNF125 had a low solubility and stability and precipitated immediately under the solution conditions tested. 15N-1H HSQC spectrum of RIG-I CARDs indicated that under the NMR condition used, free domains did not fold into a stable conformation. 160

The SARS-CoV-2 main protease of (Mpro) is an important target for SARS-CoV-2 related drug repurposing and development studies. Here, we describe the steps for structural characterization of SARS-CoV-2 Mpro, starting from plasmid preparation and protein purification. We detail the steps for crystallization using the sitting drop, microbatch (under oil) approach. Finally, we cover data collection and structure determination using serial femtosecond crystallography. For complete details on the use and execution of this protocol, please refer to Durdagi et al. (2021)., Graphical abstract, The SARS-CoV-2 main protease of (Mpro) is an important target for SARS-CoV-2 related drug repurposing and development studies. Here, we describe the steps for structural characterization of SARS-CoV-2 Mpro, starting from plasmid preparation and protein purification. We detail the steps for crystallization using the sitting drop, microbatch (under oil) approach. Finally, we cover data collection and structure determination using serial femtosecond crystallography.

Communications biology 5(1), 73 (2022). doi:10.1038/s42003-021-02903-7, 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 Å resolution and the second one is a cryogenic crystal structure (Apo-Cryo) at 1.1 Å 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., Published by Springer Nature, London

Oligomerization of Pr55(Gag) is a critical step of the late stage of the HIV life cycle. It has been known that the binding of IP6, an abundant endogenous cyclitol molecule at the MA domain, has been linked to the oligomerization of Pr55(Gag). However, the exact binding site of IP6 on MA remains unknown and the structural details of this interaction are 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 GNM-based transfer entropy calculations 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 binding of Pr55(Gag) to membrane. 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 IP6 coordination of the oligomerization of outer MA and inner CA domain's 2D layers during assembly and budding., National Science Foundation (NSF) Science and Technology Centers Grant; BioXFEL; Scientific and Technological Research Council of Turkey (TÜBİTAK); 2232 International Fellowship for Outstanding Researchers Program; Grant-in-Aid for Scientific Research; Grant-in-Aid for Research Activity Start-up; Japanese Society for the Promotion of Science (JSPS) Grant-in-Aid for Scientific Research; Grant-in and Aid for JSPS Research Fellow; Japan Agency for Medical Research and Development Basis for Supporting Innovative Drug Discovery and Life Science Research; Platform for Drug Discovery, Informatics, and Structural Life Science (MEXT); Japan Agency for Medical Research and Development (AMED)

The COVID-19 pandemic has resulted in 198 million reported infections and more than 4 million deaths as of July 2021 (covid19.who.int). Research to identify effective therapies for COVID-19 includes: (1) designing a vaccine as future protection; (2) de novo drug discovery; and (3) identifying existing drugs to repurpose them as effective and immediate treatments. To assist in drug repurposing and design, we determine two apo structures of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease at ambient temperature by serial femtosecond X-ray crystallography. We employ detailed molecular simulations of selected known main protease inhibitors with the structures and compare binding modes and energies. The combined structural and molecular modeling studies not only reveal the dynamics of small molecules targeting the main protease but also provide invaluable opportunities for drug repurposing and structure-based drug design strategies against SARS-CoV-2., Graphical abstract, Durdağı et al. represent radiation damage-free high-resolution SARS-CoV-2 main protease SFX structures obtained at near-physiological temperature and performed MD simulation of apo-form proteins and three known main protease inhibitors. The structures reveal alternate conformation, while MD simulation indicates asymmetric behavior of the protein, which is invaluable information for immediate drug-repurposing studies.

Objective: to compare the effectiveness of different mask types in limiting the dispersal of coughed air. Method: the Schlieren method with a single curved mirror was used in this study. Coughed air has a slightly higher temperature than ambient air, which generates a refractive index gradient. A curved mirror with a radius of curvature of 10 m and a diameter of 60 cm was used. The spread of the cough wavefront was investigated among five subjects wearing: (1) no mask; (2) a single surgical mask; (3) a double surgical mask; (4) a cloth mask; (5) a valveless N95 mask; and (6) a valved N95 mask. Results: all mask types reduced the size of the contaminated region significantly. The percentage reduction in the cross-sectional area of the contaminated region for the same mask types on different subjects revealed by normalized data suggests that the fit of a mask plays an important role. Conclusions: no significant difference in the spread of coughed air was found between the use of a single surgical mask or a double surgical mask. Cloth masks may be effective, depending on the quality of the cloth. Valved N95 masks exclusively protect the user. The fit of a mask is an important factor to minimize the contaminated region., 2232 International Fellowship for Outstanding Researchers Program; Scientific and Technological Research Council of Turkey (TÜBİTAK)