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 cryocrystallography has never been performed in Türkiye 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, Türkiye 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.

Bridging the Gap between Gut Microbiota and Alzheimer's Disease: A Metaproteomic Approach for Biomarker Discovery in Transgenic Mice.

Alzheimer's Disease (AD) is a progressively debilitating form of dementia that affects millions of individuals worldwide. Although a vast amount of research has investigated the complex interplay between gut microbiota and neurodegeneration, the metaproteomic effects of microbiota on AD pathogenesis remain largely uncharted territory. This study aims to reveal the role of gut microbiota in AD pathogenesis, particularly regarding changes in the proteome and molecular pathways that are intricately linked to disease progression. We operated state-of-the-art Nano-Liquid Chromatography Mass Spectrometry (nLC-MS/MS) to compare the metaproteomic shifts of 3-month-old transgenic (3M-ALZ) and control (3M-ALM, Alzheimer's Littermate) mice, depicting the early onset of AD with those of 12-month-old ALZ and ALM mice displaying the late stage of AD. Combined with computational analysis, the outcomes of the gut-brain axis-focused inquiry furnish priceless knowledge regarding the intersection of gut microbiota and AD. Accordingly, our data indicate that the microbiota, proteome, and molecular changes in the intestine arise long before the manifestation of disease symptoms. Moreover, disparities exist between the normal-aged flora and the gut microbiota of late-stage AD mice, underscoring that the identified vital phyla, proteins, and pathways hold immense potential as markers for the early and late stages of AD. Our research endeavors to offer a comprehensive inquiry into the intricate interplay between gut microbiota and Alzheimer's Disease utilizing metaproteomic approaches, which have not been widely adopted in this domain. This highlights the exigency for further scientific exploration to elucidate the underlying mechanisms that govern this complex and multifaceted linkage.

Rapid and efficient ambient temperature X-ray crystal 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 min with resolution extending to 2.39 Å 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.

In vitro antifungal and antibiofilm activities of novel sulfonyl hydrazone derivatives against Candida spp.

BACKGROUND: The aim of this study was to investigate the antifungal and antibiofilm activity of the new sulfonyl hydrazones compound derived from sulphonamides. METHODS: In this study, new sulfonyl hydrazone series were synthesized via a green chemistry method. The structures of the synthesized compounds were characterized by elemental analyses and spectroscopic methods. The antifungal activities of the Anaf compounds against Candida strains under planktonic conditions were tested. The biofilm-forming ability of Candida strains was determined and the inhibitory effects of Anaf compounds on Candida biofilms compared with fluconazole were measured by MTT assay. Expression analysis of biofilm-related genes was investigated with qRT-PCR. The statistical analysis was performed using a one-way ANOVA test. CANDIDA: strains was determined and the inhibitory effects of Anaf compounds on Candida biofilms compared with fluconazole were measured by MTT assay. Expression analysis of biofilm-related genes was investigated with qRT-PCR. The statistical analysis was performed using a one-way ANOVA test. RESULTS: A total of 16 (45.7%) out of 35 Candida isolates were determined as strong biofilm producers in this study. C. albicans was the most biofilm producer, followed by C. krusei and C. lusitaniae. The Anaf compounds had a broad spectrum of activity with MIC values ranging from 4 µg/ml to 64 µg/ml. Our data indicated that the Anaf compound had a significant effect on inhibiting biofilm formation in both fluconazole-susceptible and -resistant strains. The expression levels of hypha-specific genes als3, hwp1, ece1 and sap5 were downregulated by Anaf compounds. CONCLUSIONS: Our study revealed that the Anaf compounds had antifungal activity and inhibited fungal biofilms, which may be related to the suppression of C. albicans adherence and hyphal formation. These results suggest that Anaf compounds may have therapeutic potential for the treatment and prevention of biofilm-associated Candida infections.

A Brief Atlas of Insulin.

Insulin is an essential factor for mammalian organisms: a regulator of glucose metabolism and other key signaling pathways. Insulin is also a multifunctional hormone whose absence can cause many diseases. Recombinant insulin is widely used in the treatment of diabetes. Understanding insulin, biosimilars, and biobetters from a holistic perspective will help pharmacologically user-friendly molecules design and develop personalized medicine-oriented therapeutic strategies for diabetes. Additionally, it helps to understand the underlying mechanism of other insulindependent metabolic disorders. The purpose of this atlas is to review insulin from a biotechnological, basic science, and clinical perspective, explain nearly all insulin-related disorders and their underlying molecular mechanisms, explore exogenous/recombinant production strategies of patented and research-level insulin/analogs, and highlight their mechanism of action from a structural perspective. Combined with computational analysis, comparisons of insulin and analogs also provide novel information about the structural dynamics of insulin.

Identification of New L-Heptanoylphosphatidyl Inositol Pentakisphosphate Derivatives Targeting the Interaction with HIV-1 Gag by Molecular Modelling Studies.

The HIV-1 Gag protein binds to the host cell membrane and assembles into immature particles. Then, in the course of immature virion budding, activated protease cleaves Gag into its main components: MA, CA, NC, and p6 proteins. The highly basic residues of MA predominantly interact with the acidic head of phosphatidyl-inositol-4,5-bisphosphate (PI(4,5)P2) inserted into the membrane. Our research group developed L-Heptanoylphosphatidyl Inositol Pentakisphosphate (L-HIPPO) and previously confirmed that this compound bound to the MA more strongly than PI(4,5)P2 and inositol hexakisphosphate (IP6) did. Therefore, herein we rationally designed eight new L-HIPPO derivatives based on the fact that the most changeable parts of L-HIPPO were two acyl chains. After that, we employed molecular docking for eight compounds via Maestro software using high-resolution crystal structures of MA in complex with IP6 (PDB IDs: 7E1I, 7E1J, and 7E1K), which were recently elucidated by our research group. The most promising docking scores were obtained with benzene-inserted compounds. Thus, we generated a library containing 213 new aromatic group-inserted L-HIPPO derivatives and performed the same molecular docking procedure. According to the results, we determined the nine new L-HIPPO derivatives most effectively binding to the MA with the most favorable scoring functions and pharmacokinetic properties for further exploration.

Protocol for structure determination of SARS-CoV-2 main protease at near-physiological-temperature by serial femtosecond crystallography.

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).

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 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.

Structural insights into bifunctional thaumarchaeal crotonyl-CoA hydratase and 3-hydroxypropionyl-CoA dehydratase from Nitrosopumilus maritimus.

The ammonia-oxidizing thaumarchaeal 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle is one of the most energy-efficient CO2 fixation cycles discovered thus far. The protein encoded by Nmar_1308 (from Nitrosopumilus maritimus SCM1) is a promiscuous enzyme that catalyzes two essential reactions within the thaumarchaeal 3HP/4HB cycle, functioning as both a crotonyl-CoA hydratase (CCAH) and 3-hydroxypropionyl-CoA dehydratase (3HPD). In performing both hydratase and dehydratase activities, Nmar_1308 reduces the total number of enzymes necessary for CO2 fixation in Thaumarchaeota, reducing the overall cost for biosynthesis. Here, we present the first high-resolution crystal structure of this bifunctional enzyme with key catalytic residues in the thaumarchaeal 3HP/4HB pathway.

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

Oligomerization of Pr55Gag 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 Pr55Gag. 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 Pr55Gag 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.

Near-physiological-temperature serial crystallography reveals conformations of SARS-CoV-2 main protease active site for improved drug repurposing.

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.

Delivery of lipophilic porphyrin by liposome vehicles: preparation and photodynamic therapy activity against cancer cell lines.

Porphyrin photosensitizers are mostly used components in photodynamic therapy (PDT). The poor solubility of porphyrins in aqueous medium is the problem to be solved for the in vivo applications. The delivery of photosensitizers to the tumor cells using liposome vehicles can help to overcome this problem. In this work, we have first functionalized the protoporphyrin IX with lipophilic oleylamine arms and encapsulated it into 1,2 dioleyl-sn-glycero-phosphatidylcholine (DOPC) liposomes. The appropriate sizes of liposomes are about 140 nm and have the characteristic Soret and Q band absorptions at 405 nm (Soret), 507 nm, 541 nm, 577 nm and 631 nm (Q bands), respectively. In the photodynamic activity studies, the liposomal porphyrins were irradiated with light (375 nm, 10 mW) in the presence of cancer cell lines, HeLa and AGS. We have found that both liposomal porphyrins and oleylamine conjugated porphyrins are much more effective than PpIX. This result can be attributed to the drug delivery characteristic of the liposomes which plays effective role in endocytosis. We also found that, in AGS cells, liposomal PpIX-Ole induced apoptosis more than HeLa cells under light conditions.