Browsing by Author "Eşsiz, Şebnem"

Now showing 1 - 14 of 14

Citation - WoS: 8
Citation - Scopus: 9
Antifungal Screening and in Silico Mechanistic Studies of an In-House Azole Library
(2019) Sarı, Suat; Kart, Didem; Sabuncuoğlu, Suna; Doğan, İnci Selin; Özdemir, Zeynep; Bozbey, İrem; Gencel, Melis; Eşsiz, Şebnem; Reynisson, Jóhannes; Karakurt, Arzu; Saraç, Selma; Dalkara, Sevim; Advertising; 04. Faculty of Communication; 01. Kadir Has University
Systemic Candida infections pose a serious public health problem with high morbidity and mortality. C. albicans is the major pathogen identified in candidiasis; however, non-albicans Candida spp. with antifungal resistance are now more prevalent. Azoles are first-choice antifungal drugs for candidiasis; however, they are ineffective for certain infections caused by the resistant strains. Azoles block ergosterol synthesis by inhibiting fungal CYP51, which leads to disruption of fungal membrane permeability. In this study, we screened for antifungal activity of an in-house azole library of 65 compounds to identify hit matter followed by a molecular modeling study for their CYP51 inhibition mechanism. Antifungal susceptibility tests against standard Candida spp. including C. albicans revealed derivatives 12 and 13 as highly active. Furthermore, they showed potent antibiofilm activity as well as neglectable cytotoxicity in a mouse fibroblast assay. According to molecular docking studies, 12 and 13 have the necessary binding characteristics for effective inhibition of CYP51. Finally, molecular dynamics simulations of the C. albicans CYP51 (CACYP51) homology model's catalytic site complexed with 13 were stable demonstrating excellent binding.
Citation - WoS: 5
Citation - Scopus: 7
Computational Analysis of a Zn-Bound Tris(imidazolyl) Calix[6]arene Aqua Complex: Toward Incorporating Second-Coordination Sphere Effects Into Carbonic Anhydrase Biomimetics
(Amer Chemical Soc, 2013) Koziol, Lucas; Eşsiz, Şebnem; Wong, Sergio E.; Lau, Edmond Y.; Valdez, Carlos A.; Satcher, Joe H. Jr.; Aines, Roger D.; Lightstone, Felice C.; Molecular Biology and Genetics; 05. Faculty of Engineering and Natural Sciences; 01. Kadir Has University
Molecular dynamics simulations and quantum-mechanical calculations were performed to characterize a supra-molecular tris(imidazolyl) calix[6]arene Zn2+ aqua complex as a biomimetic model for the catalyzed hydration of carbon dioxide to bicarbonate H2O + CO2 -> H+ + HCO3-. On the basis of potential-of-mean-force (PMF) calculations stable conformations had distorted 3-fold symmetry and supported either one or zero encapsulated water molecules. The conformation with an encapsulated water molecule is calculated to be lower in free energy than the conformation with an empty cavity (Delta G = 1.2 kcal/mol) and is the calculated free-energy minimum in solution. CO2 molecule partitioning into the cavity is shown to be very facile proceeding with a barrier of 1.6 kcal/mol from a weak encounter complex which stabilizes the species by about 1.0 kcal/mol. The stabilization energy of CO2 is calculated to be larger than that of H2O (Delta Delta G = 1.4 kcal/mol) suggesting that the complex will preferentially encapsulate CO2 in solution. In contrast the PMF for a bicarbonate anion entering the cavity is calculated to be repulsive in all nonbonding regions of the cavity due to the diameter of the calix[6]arene walls. Geometry optimization of the Zn-bound hydroxide complex with an encapsulated CO2 molecule showed that multiple noncovalent interactions direct the reactants into optimal position for nucleophilic addition to occur. The calixarene complex is a structural mimic of the hydrophilic/hydrophobic divide in the enzyme providing a functional effect for CO2 addition in the catalytic cycle. The results show that Zn-binding calix[6]arene scaffolds can be potential synthetic biomimetics for CO2 hydration catalysis both in terms of preferentially encapsulating CO2 from solution and by spatially fixing the reactive species inside the cavity.
Citation - Scopus: 16
Discovery of New Azoles With Potent Activity Against Candida Spp. and Candida Albicans Biofilms Through Virtual Screening
(Elsevier, 2020) Karakurt, Arzu; Kart, Didem; Öztürk, Naile; Kaynak, F. Betül; Gencel, Melis; Taşkor, Gülce; Karakurt, Arzu; Saraç, Selma; Eşsiz, Şebnem; Dalkara, Sevim; Molecular Biology and Genetics; 05. Faculty of Engineering and Natural Sciences; 01. Kadir Has University
Systemic candidiasis is a rampant bloodstream infection ofCandidaspp. andC. albicansis the majorpathogen isolated from infected humans. Azoles, the most common class of antifungals which sufferfrom increasing resistance, and especially intrinsically resistant non-albicans Candida(NAC) species, actby inhibiting fungal lanosterol 14a-demethylase (CYP51). In this study we identified a number of azolecompounds in 1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethanol/ethanone oxime ester structurethrough virtual screening using consensus scoring approach, synthesized and tested them for theirantifungal properties. We reached several hits with potent activity against azole-susceptible and azole-resistantCandidaspp. as well as biofilms ofC. albicans.5i's minimum inhibitor concentration (MIC) was0.125mg/ml againstC. albicans, 0.5mg/ml againstC. kruseiand 1mg/ml against azole-resistantC. tropicalisisolate. Considering the MIC values offluconazole against these fungi (0.5, 32 and 512mg/ml, respec-tively),5iemerged as a highly potent derivative. The minimum biofilm inhibitor concentration (MBIC) of5c,5j, and5pwere 0.5mg/ml (and5iwas 2mg/ml) againstC. albicansbiofilms, lower than that ofamphotericin B (4mg/ml), afirst-line antifungal with antibiofilm activity. In addition, the active com-pounds showed neglectable toxicity to human monocytic cell line. We further analyzed the dockingposes of the active compounds inC. albicansCYP51 (CACYP51) homology model catalytic site andidentified molecular interactions in agreement with those of known azoles with fungal CYP51s andmutagenesis studies of CACYP51. We observed the stability of CACYP51 in complex with5iin moleculardynamics simulations.©2019 Elsevier Masson SAS. All rights reserved.1. IntroductionSystemic candidiasis is a major public health issue, especiallywith immune-suppressed cases reaching high mortality rates. Themembers of the genusCandidaare the most frequently recoveredfrom human fungal infection andCandida albicans, so far, is theleading pathogen identified in nosocomial candidiasis [1]. Inaddition to increasing drug-resistant strains ofC. albicans, emer-gence of non-albicans Candidaspp. (NAC) complicate the treatmentof mycoses [2].C. tropicalisis among the NACs that show reducedsusceptibility tofirst-line antifungals reportedly leading to break-through fungemia among high-risk patients [3,4]. Also,C. kruseiisknown to be intrinsically resistant to a number of azoles includingfluconazole [5]. One of the several mechanisms of therapy-resistance is formation of biofilms, which are complex microor-ganism colonies enclosed in an exopolysaccharide matrix on bioticand non-biotic surfaces. Persistent biofilms make fungi much lesssusceptible to antifungal drugs compared to their planktonic formsfor a number of reasons [6e8]. Therefore it is essential to design*Corresponding author. Hacettepe University Faculty of Pharmacy, Departmentof Pharmaceutical Chemistry, 06100, Sihhiye, Ankara, Turkey.E-mail addresses:[email protected],[email protected](S. Sari).Contents lists available atScienceDirectEuropean Journal of Medicinal Chemistryjournal homepage:http://www.elsevier.com/locate/ejmechhttps://doi.org/10.1016/j.ejmech.2019.06.0830223-5234/©2019 Elsevier Masson SAS. All rights reserved.European Journal of Medicinal Chemistry 179 (2019) 634e648
G. Kaustophilius Lipazinin Mutasyon Çalışmaları Kullanılarak Substrat Seçiciliğinin ve Aktivitesinin Geliştirilmesi
(2025) Poyraz, Yağmur; Eşsiz, Şebnem; Molecular Biology and Genetics; 05. Faculty of Engineering and Natural Sciences; 01. Kadir Has University
Lipazlar, yağların hidrolizini katalize eden hidrolaz sınıfı enzimlerdir. Biyoteknolojide özellikle biyodizel üretimi alanında önemli bir yeri bulunmaktadır. Bu enzimler, yağların transesterifikasyon reaksiyonunu katalize ederek bitkisel veya hayvansal yağlardan biyodizel elde etmeye yardımcı olmaktadır. Fakat, biyodizel üretiminde lipaz kullanımının bir takım sorunları bulunmaktadır. Reaksiyon verimliliğinin yetersizliği, özellikle yüksek serbest yağ asidi içeriğine sahip ham yağların transesterifikasyonunda önemli bir sorundur. Ayrıca, lipazların reaksiyon hızı açısından yeterince hızlı olmama duumu söz konusudur. Bunlara ek olarak, bu enzimler çevre koşullarına duyarlıdır; uygun sıcaklık veya pH koşulları olmaması durumu enzim aktivitesini olumsuz etkileyebilirken, kararlılık sorunlarını da beraberinde getirebilmektedir. Araştırmacılar, lipazların performansını artırmak ve süreç verimliliğini iyileştirmek için sürekli olarak yenilikçi yöntemler ve teknolojiler araştırmaktadır. Enzim mutasyonu ve lipaz immobilizasyonu, bu çabada odaklanılan önemli alanlardır. Bu çalışma, Geobacillus kaustophilus termofilik lipazının yapısal ve dinamik özellikleri hakkındaki anlayışımızı geliştirmeyi ve aktivitesini, substrat seçiciliğini ve termal kararlılığını iyileştirmek için mutasyonları kullanmayı amaçlamaktadır. İdeal mutasyonları bulmak için Gklip lipazı modellenmiş ve hem yabani tip hem de mutasyonlu yapıların ligand seçiciliğini değerlendirmek için farklı uzunlukta karbon zincirli yağ asitleri ile dok çalışmaları yürütülmüştür. Daha sonra, tüm protein-ligand kompleksleri için 50 ns moleküler dinamik simülasyonları gerçekleştirilmiştir. Simülasyonların son 30 ns'si MM/GBS analizi için kullanılmıştır. 36 mutasyondan, en iyi 5 tanesi daha ileri deneysel çalışmalar için seçilmiştir.
G. Kaustophilius Lipazinin Mutasyon Çalışmaları Kullanılarak Substrat Seçiciliğinin ve Aktivitesinin Geliştirilmesi
(2025) Poyraz, Yağmur; Eşsiz, Şebnem; Molecular Biology and Genetics; 05. Faculty of Engineering and Natural Sciences; 01. Kadir Has University
Lipazlar, yağların hidrolizini katalize eden hidrolaz sınıfı enzimlerdir. Biyoteknolojide özellikle biyodizel üretimi alanında önemli bir yeri bulunmaktadır. Bu enzimler, yağların transesterifikasyon reaksiyonunu katalize ederek bitkisel veya hayvansal yağlardan biyodizel elde etmeye yardımcı olmaktadır. Fakat, biyodizel üretiminde lipaz kullanımının bir takım sorunları bulunmaktadır. Reaksiyon verimliliğinin yetersizliği, özellikle yüksek serbest yağ asidi içeriğine sahip ham yağların transesterifikasyonunda önemli bir sorundur. Ayrıca, lipazların reaksiyon hızı açısından yeterince hızlı olmama duumu söz konusudur. Bunlara ek olarak, bu enzimler çevre koşullarına duyarlıdır; uygun sıcaklık veya pH koşulları olmaması durumu enzim aktivitesini olumsuz etkileyebilirken, kararlılık sorunlarını da beraberinde getirebilmektedir. Araştırmacılar, lipazların performansını artırmak ve süreç verimliliğini iyileştirmek için sürekli olarak yenilikçi yöntemler ve teknolojiler araştırmaktadır. Enzim mutasyonu ve lipaz immobilizasyonu, bu çabada odaklanılan önemli alanlardır. Bu çalışma, Geobacillus kaustophilus termofilik lipazının yapısal ve dinamik özellikleri hakkındaki anlayışımızı geliştirmeyi ve aktivitesini, substrat seçiciliğini ve termal kararlılığını iyileştirmek için mutasyonları kullanmayı amaçlamaktadır. İdeal mutasyonları bulmak için Gklip lipazı modellenmiş ve hem yabani tip hem de mutasyonlu yapıların ligand seçiciliğini değerlendirmek için farklı uzunlukta karbon zincirli yağ asitleri ile dok çalışmaları yürütülmüştür. Daha sonra, tüm protein-ligand kompleksleri için 50 ns moleküler dinamik simülasyonları gerçekleştirilmiştir. Simülasyonların son 30 ns'si MM/GBS analizi için kullanılmıştır. 36 mutasyondan, en iyi 5 tanesi daha ileri deneysel çalışmalar için seçilmiştir.
Loop Modeling and Molecular Dynamics Simulations of Apo and Ligand-Bound Human Glun1-Hlun2a Nmda Type Receptors
(Kadir Has Üniversitesi, 2017) Aktolun, Muhammed; Eşsiz, Şebnem; Molecular Biology and Genetics; 05. Faculty of Engineering and Natural Sciences; 01. Kadir Has University
N-Methyl-D-Aspartate receptors (NMDARs) are glutamate-gated ion channels found in the nerve cell membranes. The functioning of the receptor is of crucial importance in consciousness and normal brain functions. As a result of overexcitation of NMDARs neuronal death occurs and may lead to diseases such as epilepsy stroke Alzheimer's and Parkinson's. Understanding the molecular mechanism and structure function relationships of the receptor might lead to discovery of new drug target mechanisms. Recently there are two intact X-ray structures available one is from Xenopus laevis and the other one is from Rattus norvegicus for GluN1-GluN2B type NMDA receptor. First both Xray structures are examined and compared for the ion channel especially by taking the general problems into consideration which arise from crystallization conditions. Human GluN1- GluN2A type NMDAR structure is modeled based on the structure of Xenopus laevis template and missing loops are added by ab-initio loop modeling. Final structure is chosen according to the model assessment scoring function. NMDAR activation requires binding of two coagonists glycine and glutamate. To be able to observe the structural changes upon ligand binding glycine and glutamate molecules are docked into the corresponding binding sites of the receptor. Subsequently Molecular Dynamics (MD) simulations of 1 microsecond are performed for both apo and ligand-bound structures. 10 structural parameters which have been considered as functionally important in previous NMDA studies are developed to understand the dynamics of the conformational changes that is associated with the function of the protein throughout the simulations. Moreover Principal Component Analysis is performed for the equilibrated part of the simulations to classify similar conformations together. in the ligand-bound simulation certain loop regions showed higher mobility. Upon ligand binding closure in LBD clamshell smaller ATD-LBD inter-domain distance and larger LBDTMD linker distance is observed in specific subunits. Opening in the bottom TMD girdle is observed for a short time. Correlated motions of the receptor in the ligand-bound simulation increased. The structure showed rotation-like motion in the apo simulation whereas slidinglike motion within the neighboring heterodimers are observed.
Modelling of C-Terminal Tail of Human Sting and Its Interaction With\rtank-Binding Kinase 1
(2022) Masrı, Rahaf Ata Ouda Al; Bıda, Hajara Audu; Eşsiz, Şebnem; Molecular Biology and Genetics; 05. Faculty of Engineering and Natural Sciences; 01. Kadir Has University
Stimulator of interferon genes (STING) plays a significant role in a cell’s intracellular defense against pathogens or selfDNA by inducing inflammation or apoptosis through a pathway known as cGAS-cGAMP-STING. STING uses one of its domains, the\rC-terminal tail (CTT) to recruit the members of the pathway. However, the structure of this domain has not been solved experimentally.\rSTING conformation is open and more flexible when inactive. When STING gets activated by cGAMP, its conformation changes to a\rclosed state covered by 4 beta-sheets over the binding site. This conformational change leads to its binding to Tank-binding kinase 1\r(TBK1). TBK1 then phosphorylates STING aiding its entry to the cell’s nucleus.\rIn this study, we focused on the loop modeling of the CTT domain in both the active and inactive STING conformations. After the\rmodeling step, the active and inactive STING structures were docked to one of the cGAS-cGAMP-STING pathway members, TBK1,\rto observe the differences of binding modes. CTT loop stayed higher in the active structure, while all the best-scored models, active or\rinactive, ended up around the same position with respect to TBK1. However, when the STING poses are compared with the cryo-EM\rimage of the complex structure, the models in the active structure chain B displayed closer results to the complex structure.
Citation - WoS: 3
Citation - Scopus: 3
The Neural Gamma(2)alpha(1)beta(2)alpha(1)beta(2) Gamma Amino Butyric Acid Ion Channel Receptor: Structural Analysis of the Effects of the Ivermectin Molecule and Disulfide Bridges
(Springer, 2018) Ayan, Meral; Eşsiz, Şebnem; Molecular Biology and Genetics; 05. Faculty of Engineering and Natural Sciences; 01. Kadir Has University
While similar to 30% of the human genome encodes membrane proteins only a handful of structures of membrane proteins have been resolved to high resolution. Here we studied the structure of a member of the Cys-loop ligand gated ion channel protein superfamily of receptors human type A gamma(2)alpha(1)beta(2)alpha(1)beta(2) gamma amino butyric acid receptor complex in a lipid bilayer environment. Studying the correlation between the structure and function of the gamma amino butyric acid receptor may enhance our understanding of the molecular basis of ion channel dysfunctions linked with epilepsy ataxia migraine schizophrenia and other neurodegenerative diseases. The structure of human gamma(2)alpha(1)beta(2)alpha(1)beta(2) has been modeled based on the X-ray structure of the Caenorhabditis elegans glutamate-gated chloride channel via homology modeling. The template provided the first inhibitory channel structure for the Cys-loop superfamily of ligand-gated ion channels. The only available template structure before this glutamate-gated chloride channel was a cation selective channel which had very low sequence identity with gamma aminobutyric acid receptor. Here our aim was to study the effect of structural corrections originating from modeling on a more reliable template structure. The homology model was analyzed for structural properties via a 100 ns molecular dynamics (MD) study. Due to the structural shifts and the removal of an open channel potentiator molecule ivermectin from the template structure helical packing changes were observed in the transmembrane segment. Namely removal of ivermectin molecule caused a closure around the Leu 9 position along the ion channel. In terms of the structural shifts there are three potential disulfide bridges between the M1 and M3 helices of the gamma(2) and 2 alpha(1) subunits in the model. The effect of these disulfide bridges was investigated via monitoring the differences in root mean square fluctuations (RMSF) of individual amino acids and principal component analysis of the MD trajectory of the two homology models-one with the disulfide bridge and one with protonated Cys residues. In all subunit types RMSF of the transmembrane domain helices are reduced in the presence of disulfide bridges. Additionally loop A loop F and loop C fluctuations were affected in the extracellular domain. In cross-correlation analysis of the trajectory the two model structures displayed different coupling in between the M2-M3 linker region protruding from the membrane and the beta 1-beta 2/D loop and cys-loop regions in the extracellular domain. Correlations of the C loop which collapses directly over the bound ligand molecule were also affected by differences in the packing of transmembrane helices. Finally more localized correlations were observed in the transmembrane helices when disulfide bridges were present in the model. The differences observed in this study suggest that dynamic coupling at the interface of extracellular and ion channel domains differs from the coupling introduced by disulfide bridges in the transmembrane region. We hope that this hypothesis will be tested experimentally in the near future.
Citation - WoS: 49
Citation - Scopus: 53
New Azole Derivatives Showing Antimicrobial Effects and Their Mechanism of Antifungal Activity by Molecular Modeling Studies
(Elsevier France-Editions Scientifiques Medicales Elsevier, 2017) Doğan, İnci Selin; Saraç, Selma; Sarı, Suat; Kart, Didem; Eşsiz, Şebnem; Vural, İmran; Dalkara, Sevim; Molecular Biology and Genetics; 05. Faculty of Engineering and Natural Sciences; 01. Kadir Has University
Azole antifungals are potent inhibitors of fungal lanosterol 14 alpha demethylase (CYP51) and have been used for eradication of systemic candidiasis clinically. Herein we report the design synthesis and biological evaluation of a series of 1-phenyl/1-(4-chlorophenyl)-2-(1H-imidazol-1-yl) ethanol esters. Many of these derivatives showed fungal growth inhibition at very low concentrations. Minimal inhibition concentration (MIC) value of 15 was 0.125 mu g/mL against Candida albicans. Additionally some of our compounds such as 19 (MIC: 0.25 mu g/mL) were potent against resistant C. glabrata a fungal strain less susceptible to some first-line antifungal drugs. We confirmed their antifungal efficacy by antibiofilm test and their safety against human monocytes by cytotoxicity assay. To rationalize their mechanism of action we performed computational analysis utilizing molecular docking and dynamics simulations on the C. albicans and C. glabrata CYP51 (CACYP51 and CGCYP51) homology models we built. Leu130 and T131 emerged as possible key residues for inhibition of CGCYP51 by 19. (C) 2017 Elsevier Masson SAS. All rights reserved.
Soman as a Wrench in the Works of Human Acetylcholinesterase: Soman Induced Conformational Changes Revealed by Molecular Dynamics Simulations
(Amer Chemical Soc, 2014) Bennion, Brian J.; Eşsiz, Şebnem; Lau, Edmond Y.; Fattebert, Jean-Luc; Emigh, Aiyana; Lightstone, Felice C.; Molecular Biology and Genetics; 05. Faculty of Engineering and Natural Sciences; 01. Kadir Has University
[Abstract Not Available]
Structural, Optical and Antibacterial Properties of Ws2 Doped Zno Nanoparticles
(2023) Beytür, Sercan; Uysal, Bengü Özuğur; Eşsiz, Şebnem; Molecular Biology and Genetics; 05. Faculty of Engineering and Natural Sciences; 01. Kadir Has University
Yapısal ve optik özelliklerinin yanı sıra antibakteriyel özellikleri ile tanınan ZnO nanoparçacıkları, çeşitli alanlarda yaygın olarak uygulanmaktadır. Metaller veya metal oksitler gibi farklı malzemelerin ZnO'ya katkılanmasının özelliklerini iyileştirdiği bilinmektedir. Burada %5, %15 ve %25 oranlarında WS2 katkılı ZnO'dan oluşan nanofilmler sentezlenmekte ve özellikleri araştırılmaktadır. Moleküler yerleştirme analizleri ile desteklenen, farklı oranlarda WS2 ilave edildikten sonra bakterisidal özelliklerin arttırılması vurgulanmaktadır ve ilgili proteinlerin hedeflenmesi yoluyla bakteriyel hayatta kalmada çok önemli bir rol oynayan kalıntıların inhibe edici etkileşimi tarafından desteklenmektedir.
Citation - WoS: 54
Citation - Scopus: 62
Synthesis, Biological Evaluation and Molecular Docking Studies of Bis-Chalcone Derivatives as Xanthine Oxidase Inhibitors and Anticancer Agents
(Elsevier, 2019) Burmaoğlu, Serdar; Özcan, Şeyda; Balcıoğlu, Sevgi; Gencel, Melis; Noma, Samir Abbas Ali; Eşsiz, Şebnem; Ateş, Burhan; Algül, Öztekin; Molecular Biology and Genetics; 05. Faculty of Engineering and Natural Sciences; 01. Kadir Has University
In this study, a series of B-ring fluoro substituted bis-chalcone derivatives were synthesized by Claisen-Schmidt condensation reactions and evaluated for their ability to inhibit xanthine oxidase (XO) and growth inhibitory activity against MCF-7 and Caco-2 human cancer cell lines, in vitro. According to the results obtained, the bis-chalcone with fluoro group at the 2 (4b) or 2,5-position (4g) of B-ring were found to be potent inhibitors of the enzyme with IC50 values in the low micromolar range. The effects of these compounds were about 7 fold higher than allopurinol. The binding modes of the bis-chalcone derivatives in the active site of xanthine oxidase were explained using molecular docking calculations. Also, compound 4g and 4h showed in vitro growth inhibitory activity against a panel of two human cancer cell lines 1.9 and 6.8 μM of IC50 values, respectively.
Citation - WoS: 31
Citation - Scopus: 32
A Wrench in the Works of Human Acetylcholinesterase: Soman Induced Conformational Changes Revealed by Molecular Dynamics Simulations
(Public Library Science, 2015) Bennion, Brian J.; Eşsiz, Şebnem; Lau, Edmond Y.; Fattebert, Jean-Luc; Emigh, Aiyana; Lightstone, Felice C.; Molecular Biology and Genetics; 05. Faculty of Engineering and Natural Sciences; 01. Kadir Has University
Irreversible inactivation of human acetylcholinesterase (hAChE) by organophosphorous pesticides (OPs) and chemical weapon agents (CWA) has severe morbidity and mortality consequences. We present data from quantum mechanics/molecular mechanics (QM/MM) and 80 classical molecular dynamics (MD) simulations of the apo and soman-adducted forms of hAChE to investigate the effects on the dynamics and protein structure when the catalytic Serine 203 is phosphonylated. We find that the soman phosphonylation of the active site Ser203 follows a water assisted addition-elimination mechanism with the elimination of the fluoride ion being the highest energy barrier at 6.5 kcal/mole. We observe soman-dependent changes in backbone and sidechain motions compared to the apo form of the protein. These alterations restrict the soman-adducted hAChE to a structural state that is primed for the soman adduct to be cleaved and removed from the active site. The altered motions and resulting structures provide alternative pathways into and out of the hAChE active site. In the soman-adducted protein both side and back door pathways are viable for soman adduct access. Correlation analysis of the apo and soman adducted MD trajectories shows that the correlation of gorge entrance and back door motion is disrupted when hAChE is adducted. This supports the hypothesis that substrate and product can use two different pathways as entry and exit sites in the apo form of the protein. These alternative pathways have important implications for the rational design of medical countermeasures.
Yapısal ve Mutasyonel Çalışmalar: Nmda Tipi Glutamat Reseptörü ve Format Dehidrogenaz Enziminin Moleküler Dinamik Simülasyonları
(2023) Servili, Burak; Eşsiz, Şebnem; Core Program; Molecular Biology and Genetics; 05. Faculty of Engineering and Natural Sciences; 07. Core Program; 01. Kadir Has University
Yapısal biyoloji ve moleküler dinamik (MD) simülasyon çalışmaları, moleküler düzeyde moleküller arası/içi etkileşimleri anlamanın temellerinden, ilaç ve protein tasarımının önde gelen yöntemlerinden biridir. Bu tez, iki farklı alandaki protein simülasyonlarından oluşmaktadır: Sinir hücrelerinde elektrik sinyali yayılımında bir iyon kanalı reseptör kompleksi olan N-metil-D-aspartat reseptörü (NMDAR) ve formatın bikarbonata biyokatalizinde format dehidrojenaz enzimi (FDH). Tezin ilk amacı NMDAR iyon kanalının açık yapısını mutasyonlarla elde etmektir. İkinci amaç ise allosterik bölgede Asp188Arg mutasyonu yaparak FDH enziminin hem format hem de bikarbonat substratları ile etkileşimlerini anlamaktır. Bu mutasyon deneysel olarak FDH'nin bikarbonat/CO2 yakalama için kullanımı yönünde olan ters reaksiyon için daha aktif bir enzim yaratmıştır. Bu amaçla, nano ölçekli moleküler dinamik (NAMD) uygulaması, kök-ortalama-kare sapması, kök-ortalama-kare dalgalanması ve protein-ligand etkileşim analizi yöntemleriyle birlikte simülasyonları çalıştırmak için kullanılmıştır. İlk durumda, NMDAR iyon kanalının üst kapısındaki Lurcher motifindeki alanin (A7), deneysel çalışmalara dayanarak arginin/tirozin ile değiştirildi. Analiz sonucunda NMDAR iyon kanalının açık yapısı elde edildi. FDH'nin MD simülasyonlarında, bikarbonat bağlı yapı, enzimin subtrat ve koenzim bağlanma bölgelerini ayıran önemli bir tuz köprüsünü korumuştur. Ek olarak, bağlanma bölgesinden substrat taşınımı, vahşi tip ve mutasyona uğramış yapılar için farklı yollar sergilemiştir. Her iki protein sistemi için de MD, harici pertürbasyonların proteinlerin yapısı ve işlevi, yani işlevsel mutasyonlar üzerindeki etkisini incelemek için ana araç olarak kullanılmıştır. Her iki protein de hareketlerin zaman ölçeği, sistem boyutu ve istenen ters reaksiyon yönünde düşük enzim aktivitesi nedeniyle yapısal bir çalışma için karmaşık sistemler oluşturmuştur.