Pub Date : 2024-06-02DOI: 10.1007/s10930-024-10205-0
Md Harun Rashid, Priyankar sen
Modern medicine has increased the human lifespan. However, with an increase in average lifespan risk of amyloidosis increases. Amyloidosis is a condition characterized by protein misfolding and aggregation. Early detection of amyloidosis is crucial, yet conventional diagnostic methods are costly and lack precision, necessitating innovative tools. This review explores recent advancements in diverse amyloid detection methodologies, highlighting the need for interdisciplinary research to develop a miniaturized electrochemical biosensor leveraging nanotechnology. However, the diagnostics industry faces obstacles such as skilled labor shortages, standardized selection processes, and concurrent multi-analyte identification challenges. Research efforts are focused on integrating electrochemical techniques into clinical applications and diagnostics, with the successful transition of miniaturized technologies from development to testing posing a significant hurdle. Label-free transduction techniques like voltammetry and electrochemical impedance spectroscopy (EIS) have gained traction due to their rapid, cost-effective, and user-friendly nature.
{"title":"Recent Advancements in Biosensors for the Detection and Characterization of Amyloids: A Review","authors":"Md Harun Rashid, Priyankar sen","doi":"10.1007/s10930-024-10205-0","DOIUrl":"10.1007/s10930-024-10205-0","url":null,"abstract":"<div><p>Modern medicine has increased the human lifespan. However, with an increase in average lifespan risk of amyloidosis increases. Amyloidosis is a condition characterized by protein misfolding and aggregation. Early detection of amyloidosis is crucial, yet conventional diagnostic methods are costly and lack precision, necessitating innovative tools. This review explores recent advancements in diverse amyloid detection methodologies, highlighting the need for interdisciplinary research to develop a miniaturized electrochemical biosensor leveraging nanotechnology. However, the diagnostics industry faces obstacles such as skilled labor shortages, standardized selection processes, and concurrent multi-analyte identification challenges. Research efforts are focused on integrating electrochemical techniques into clinical applications and diagnostics, with the successful transition of miniaturized technologies from development to testing posing a significant hurdle. Label-free transduction techniques like voltammetry and electrochemical impedance spectroscopy (EIS) have gained traction due to their rapid, cost-effective, and user-friendly nature.</p></div>","PeriodicalId":793,"journal":{"name":"The Protein Journal","volume":null,"pages":null},"PeriodicalIF":1.9,"publicationDate":"2024-06-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141187230","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-06-02DOI: 10.1007/s10930-024-10207-y
Yakov I. Mokin, Olga I. Povarova, Iuliia A. Antifeeva, Alexey V. Artemov, Vladimir N. Uversky, Konstantin K. Turoverov, Irina M. Kuznetsova, Alexander V. Fonin
Actin is present in the cytoplasm and nucleus of every eukaryotic cell. In the cytoplasm, framework and motor functions of actin are associated with its ability to polymerize to form F-actin. In the nucleus, globular actin plays a significant functional role. For a globular protein, actin has a uniquely large number of proteins with which it interacts. Bioinformatics analysis of the actin interactome showed that only a part of actin-binding proteins are both cytoplasmic and nuclear. There are proteins that interact only with cytoplasmic, or only with nuclear actin. The first pool includes proteins associated with the formation, regulation, and functioning of the actin cytoskeleton predominate, while nuclear actin-binding proteins are involved in the majority of key nuclear processes, from regulation of transcription to DNA damage response. Bioinformatics analysis of the structure of actin-binding proteins showed that these are mainly intrinsically disordered proteins, many of which are part of membrane-less organelles. Interestingly, although the number of intrinsically disordered actin-binding proteins in the nucleus is greater than in the cytoplasm, the drivers for the formation of the membrane-less organelles in the cytoplasm are significantly (four times) greater than in the nucleus.
肌动蛋白存在于每一个真核细胞的细胞质和细胞核中。在细胞质中,肌动蛋白的框架和运动功能与其聚合形成 F-肌动蛋白的能力有关。在细胞核中,球状肌动蛋白发挥着重要的功能作用。对于球状蛋白质来说,肌动蛋白与之相互作用的蛋白质数量之多是独一无二的。对肌动蛋白相互作用组的生物信息学分析表明,只有一部分肌动蛋白结合蛋白既与细胞质结合,又与细胞核结合。有些蛋白质只与细胞质肌动蛋白相互作用,有些则只与核肌动蛋白相互作用。第一类蛋白主要包括与肌动蛋白细胞骨架的形成、调节和功能有关的蛋白,而核肌动蛋白结合蛋白则参与了从转录调节到 DNA 损伤反应等大多数关键的核过程。对肌动蛋白结合蛋白结构的生物信息学分析表明,这些蛋白主要是内在无序蛋白,其中许多是无膜细胞器的一部分。有趣的是,虽然细胞核中本质无序的肌动蛋白结合蛋白的数量比细胞质中的多,但细胞质中形成无膜细胞器的驱动力却比细胞核中的大得多(四倍)。
{"title":"Bioinformatics Analysis of Actin Interactome: Characterization of the Nuclear and Cytoplasmic Actin-Binding Proteins","authors":"Yakov I. Mokin, Olga I. Povarova, Iuliia A. Antifeeva, Alexey V. Artemov, Vladimir N. Uversky, Konstantin K. Turoverov, Irina M. Kuznetsova, Alexander V. Fonin","doi":"10.1007/s10930-024-10207-y","DOIUrl":"10.1007/s10930-024-10207-y","url":null,"abstract":"<div><p>Actin is present in the cytoplasm and nucleus of every eukaryotic cell. In the cytoplasm, framework and motor functions of actin are associated with its ability to polymerize to form F-actin. In the nucleus, globular actin plays a significant functional role. For a globular protein, actin has a uniquely large number of proteins with which it interacts. Bioinformatics analysis of the actin interactome showed that only a part of actin-binding proteins are both cytoplasmic and nuclear. There are proteins that interact only with cytoplasmic, or only with nuclear actin. The first pool includes proteins associated with the formation, regulation, and functioning of the actin cytoskeleton predominate, while nuclear actin-binding proteins are involved in the majority of key nuclear processes, from regulation of transcription to DNA damage response. Bioinformatics analysis of the structure of actin-binding proteins showed that these are mainly intrinsically disordered proteins, many of which are part of membrane-less organelles. Interestingly, although the number of intrinsically disordered actin-binding proteins in the nucleus is greater than in the cytoplasm, the drivers for the formation of the membrane-less organelles in the cytoplasm are significantly (four times) greater than in the nucleus.</p></div>","PeriodicalId":793,"journal":{"name":"The Protein Journal","volume":null,"pages":null},"PeriodicalIF":1.9,"publicationDate":"2024-06-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141187225","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Renilla luciferase catalyzes the oxidation of coelenterazine to coelenteramide and results in the emission of a photon of light. Although Renilla luciferase has various applications in biotechnology, its low thermal stability limits the development of its applications. Arginine is a well-known stabilizing amino acid that plays a key role in protein stabilization against inactivation. However, its impact on enzyme properties is unpredictable. This study investigates the impact of arginine on the kinetics and thermal stability of Renilla luciferase. The enzyme's performance was significantly enhanced in the presence of arginine, with catalytic efficiency increasing by 3.31-fold and 3.08-fold when exposed to 0.2 M and 0.3 M arginine, respectively. Additionally, arginine improved the thermal stability of Renilla luciferase. Molecular dynamics simulation showed that the addition of 0.2 M arginine reduced the binding of coelenteramide, the reaction product and an enzyme inhibitor, to the active site of the Renilla luciferase. Therefore, the release of the product was accelerated, and the affinity of Renilla luciferase for coelenterazine increased. Furthermore, Molecular dynamics studies indicated an increased network of water molecules surrounding Renilla luciferase in the presence of 0.2 M arginine. This network potentially enhances the hydrophobic effect on the protein structure, ultimately improving enzyme stability. The findings of this study hold promise for the development of commercial kits incorporating Renilla luciferase.
瑞宁拉荧光素酶催化腔肠素氧化成腔肠酰胺,从而发出光子。尽管瑞宁拉荧光素酶在生物技术中有多种应用,但其热稳定性较低,限制了其应用的发展。精氨酸是一种众所周知的稳定氨基酸,在稳定蛋白质防止失活方面起着关键作用。然而,精氨酸对酶特性的影响却难以预测。本研究探讨了精氨酸对雷尼拉荧光素酶动力学和热稳定性的影响。在精氨酸存在的情况下,酶的性能显著提高,当暴露于 0.2 M 和 0.3 M 精氨酸时,催化效率分别提高了 3.31 倍和 3.08 倍。此外,精氨酸还提高了雷尼拉荧光素酶的热稳定性。分子动力学模拟显示,加入 0.2 M 精氨酸后,反应产物和酶抑制剂腔肠酰胺与雷尼拉荧光素酶活性位点的结合减少。因此,产物的释放速度加快,而雷尼拉荧光素酶对腔肠素的亲和力增加。此外,分子动力学研究表明,在 0.2 M 精氨酸存在的情况下,Renilla 荧光素酶周围的水分子网络增加。这种网络可能会增强蛋白质结构的疏水效应,最终提高酶的稳定性。这项研究的结果为开发含有雷尼拉荧光素酶的商业试剂盒带来了希望。
{"title":"Exploring the Potential of Arginine to Increase Coelenterazine-Renilla Luciferase Affinity and Enzyme Stability: Kinetic and Molecular Dynamics Studies","authors":"Maryam Salehian, Rahman Emamzadeh, Mahboobeh Nazari","doi":"10.1007/s10930-024-10208-x","DOIUrl":"10.1007/s10930-024-10208-x","url":null,"abstract":"<div><p><i>Renilla</i> luciferase catalyzes the oxidation of coelenterazine to coelenteramide and results in the emission of a photon of light. Although <i>Renilla</i> luciferase has various applications in biotechnology, its low thermal stability limits the development of its applications. Arginine is a well-known stabilizing amino acid that plays a key role in protein stabilization against inactivation. However, its impact on enzyme properties is unpredictable. This study investigates the impact of arginine on the kinetics and thermal stability of <i>Renilla</i> luciferase. The enzyme's performance was significantly enhanced in the presence of arginine, with catalytic efficiency increasing by 3.31-fold and 3.08-fold when exposed to 0.2 M and 0.3 M arginine, respectively. Additionally, arginine improved the thermal stability of <i>Renilla</i> luciferase. Molecular dynamics simulation showed that the addition of 0.2 M arginine reduced the binding of coelenteramide, the reaction product and an enzyme inhibitor, to the active site of the <i>Renilla</i> luciferase. Therefore, the release of the product was accelerated, and the affinity of <i>Renilla</i> luciferase for coelenterazine increased. Furthermore, Molecular dynamics studies indicated an increased network of water molecules surrounding <i>Renilla</i> luciferase in the presence of 0.2 M arginine. This network potentially enhances the hydrophobic effect on the protein structure, ultimately improving enzyme stability. The findings of this study hold promise for the development of commercial kits incorporating <i>Renilla</i> luciferase.</p></div>","PeriodicalId":793,"journal":{"name":"The Protein Journal","volume":null,"pages":null},"PeriodicalIF":1.9,"publicationDate":"2024-06-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141187227","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-20DOI: 10.1007/s10930-024-10206-z
Sathya Moorthy Ponnuraj, Neelagandan Kamariah, Balasubramanian Moovarkumudalvan, Ramya Ramadoss, M. N. Ponnuswamy
Hemoglobin (Hb) is the key metalloprotein within red blood cells involved in oxygen transportation from lungs to body cells. The heme-iron atom inherent within Hb effectuates the mechanism of oxygen transportation and carbon dioxide removal. Structural investigations on avian Hb are limited when compared with the enormous work has been carried out on mammalian Hb. Here, the crystal structure of T-state methemoglobin (T-metHb) from domestic duck (Anas platyrhynchos), a low oxygen affinity avian species, determined to 2.1Å resolution is presented. Duck T-metHb crystallized in the orthorhombic space group C2221 with unit cell parameters a = 59.89, b = 109.42 and c = 92.07Å. The final refined model with R-factor: 19.5% and Rfree: 25.2% was obtained. The structural analysis reveals that duck T-metHb adopts a unique quaternary structure that is distinct from any of the avian liganded Hb structures. Moreover, it closely resembles the deoxy Hb of bar-headed goose, a high oxygen-affinity species. Besides the amino acid αPro119 located in the α1β1 interface, a unique quaternary structure with a constrained heme environment is attributed for the intrinsic low oxygen-affinity of duck Hb. This study reports the first protein crystal structure of low oxygen-affinity avian T-metHb from Anas platyrhynchos.
血红蛋白(Hb)是红细胞内的关键金属蛋白,参与从肺到身体细胞的氧气运输。Hb 中固有的血红素-铁原子实现了氧气运输和二氧化碳清除的机制。与哺乳动物 Hb 的大量研究工作相比,对鸟类 Hb 的结构研究十分有限。本文展示了家鸭(Anas platyrhynchos)这种低氧亲和力禽类的 T 态高铁血红蛋白(T-metHb)的晶体结构,其分辨率为 2.1 Å。鸭 T-metHb 在正交空间群 C2221 中结晶,单胞参数 a = 59.89、b = 109.42 和 c = 92.07 Å。最终得到的精炼模型的 R 因子为 19.5%,Rfree 为 25.2%:25.2%。结构分析表明,鸭 T-metHb 采用了独特的四元结构,不同于任何禽类配体 Hb 结构。此外,它与棒头鹅的脱氧 Hb(一种高氧亲和性物种)非常相似。除了位于α1β1界面的氨基酸αPro119外,鸭血红蛋白具有独特的四元结构和受约束的血红素环境,这也是鸭血红蛋白固有的低氧亲和性的原因。本研究首次报道了鸭血红蛋白(Anas platyrhynchos)的低亲氧性禽 T-metHb 蛋白晶体结构。
{"title":"Molecular Insights of an Avian Species with Low Oxygen Affinity, the Crystal Structure of Duck T-State Methemoglobin","authors":"Sathya Moorthy Ponnuraj, Neelagandan Kamariah, Balasubramanian Moovarkumudalvan, Ramya Ramadoss, M. N. Ponnuswamy","doi":"10.1007/s10930-024-10206-z","DOIUrl":"10.1007/s10930-024-10206-z","url":null,"abstract":"<div><p>Hemoglobin (Hb) is the key metalloprotein within red blood cells involved in oxygen transportation from lungs to body cells. The heme-iron atom inherent within Hb effectuates the mechanism of oxygen transportation and carbon dioxide removal. Structural investigations on avian Hb are limited when compared with the enormous work has been carried out on mammalian Hb. Here, the crystal structure of T-state methemoglobin (T-metHb) from domestic duck (<i>Anas platyrhynchos</i>), a low oxygen affinity avian species, determined to 2.1Å resolution is presented. Duck T-metHb crystallized in the orthorhombic space group C222<sub>1</sub> with unit cell parameters a = 59.89, b = 109.42 and c = 92.07Å. The final refined model with R-factor: 19.5% and R<sub>free</sub>: 25.2% was obtained. The structural analysis reveals that duck T-metHb adopts a unique quaternary structure that is distinct from any of the avian liganded Hb structures. Moreover, it closely resembles the deoxy Hb of bar-headed goose, a high oxygen-affinity species. Besides the amino acid αPro119 located in the α1β1 interface, a unique quaternary structure with a constrained heme environment is attributed for the intrinsic low oxygen-affinity of duck Hb. This study reports the first protein crystal structure of low oxygen-affinity avian T-metHb from <i>Anas platyrhynchos</i>.</p></div>","PeriodicalId":793,"journal":{"name":"The Protein Journal","volume":null,"pages":null},"PeriodicalIF":1.9,"publicationDate":"2024-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141066347","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-17DOI: 10.1007/s10930-024-10203-2
Amir Sajjad Hojjati-Razgi, Shahram Nazarian, Hossein Samiei-Abianeh, Amir Vazirizadeh, Emad kordbacheh, Seyed Mojtaba Aghaie
Stonustoxin (SNTX) is a lethal protein found in stonefish venom, responsible for many of the symptoms associated with stonefish envenomation. To counter stonefish venom challenges, antivenom is a well-established and effective solution. In this study, we aimed to produce the recombinant alpha subunit protein of Stonustoxin from Synanceia horrida and prepare antibodies against it The SNTXα gene sequence was optimized for E. coli BL21 (DE3) expression and cloned into the pET17b vector. Following purification, the recombinant protein was subcutaneously injected into rabbits, and antibodies were extracted from rabbit´s serum using a G protein column As a result of codon optimization, the codon adaptation index for the SNTXα cassette increased to 0.94. SDS-PAGE analysis validated the expression of SNTXα, with a band observed at 73.5 kDa with a yield of 60 mg/l. ELISA results demonstrated rabbits antibody titers were detectable up to a 1:256,000 dilution. The isolated antibody from rabbit´s serum exhibited a concentration of 1.5 mg/ml, and its sensitivity allowed the detection of a minimum protein concentration of 9.7 ng. In the neutralization assay the purified antibody against SNTXα protected mice challenged with 2 LD50. In conclusion, our study successfully expressed the alpha subunit of Stonustoxin in a prokaryotic host, enabling the production of antibodies for potential use in developing stonefish antivenom.