首页 > 最新文献

Biophysical chemistry最新文献

英文 中文
The Drosophila RNA binding protein Hrp48 binds a specific RNA sequence of the msl-2 mRNA 3’ UTR to regulate translation 果蝇 RNA 结合蛋白 Hrp48 与 msl-2 mRNA 3' UTR 的特定 RNA 序列结合,从而调节翻译。
IF 3.3 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-10-29 DOI: 10.1016/j.bpc.2024.107346
Repression of msl-2 mRNA translation is essential for viability of Drosophila melanogaster females to prevent hypertranscription of both X chromosomes. This translational control event is coordinated by the female-specific protein Sex-lethal (Sxl) which recruits the RNA binding proteins Unr and Hrp48 to the 3’ untranslated region (UTR) of the msl-2 transcript and represses translation initiation. The mechanism exerted by Hrp48 during translation repression and its interaction with msl-2 are not well understood. Here we investigate the RNA binding specificity and affinity of the tandem RNA recognition motifs of Hrp48. Using NMR spectroscopy, molecular dynamics simulations and isothermal titration calorimetry, we identified the exact region of msl-2 3’ UTR recognized by Hrp48. Additional biophysical experiments and translation assays give further insights into complex formation of Hrp48, Unr, Sxl and RNA. Our results show that Hrp48 binds independent of Sxl and Unr downstream of the E and F binding sites of Sxl and Unr to msl-2.
抑制msl-2 mRNA的翻译对黑腹果蝇雌体的存活至关重要,以防止两条X染色体的过度转录。这种翻译控制事件由雌性特异性蛋白Sex-lethal(Sxl)协调,Sxl将RNA结合蛋白Unr和Hrp48招募到msl-2转录本的3'非翻译区(UTR),并抑制翻译启动。Hrp48在翻译抑制过程中的作用机制及其与msl-2的相互作用尚不十分清楚。在这里,我们研究了 Hrp48 的串联 RNA 识别基团的 RNA 结合特异性和亲和性。利用核磁共振光谱、分子动力学模拟和等温滴定量热法,我们确定了 Hrp48 识别的 msl-2 3' UTR 的确切区域。其他生物物理实验和翻译测定进一步揭示了 Hrp48、Unr、Sxl 和 RNA 形成复合物的情况。我们的结果表明,Hrp48与msl-2的Sxl和Unr的E和F结合位点下游结合,而不依赖于Sxl和Unr。
{"title":"The Drosophila RNA binding protein Hrp48 binds a specific RNA sequence of the msl-2 mRNA 3’ UTR to regulate translation","authors":"","doi":"10.1016/j.bpc.2024.107346","DOIUrl":"10.1016/j.bpc.2024.107346","url":null,"abstract":"<div><div>Repression of <em>msl-2</em> mRNA translation is essential for viability of <em>Drosophila melanogaster</em> females to prevent hypertranscription of both X chromosomes. This translational control event is coordinated by the female-specific protein Sex-lethal (Sxl) which recruits the RNA binding proteins Unr and Hrp48 to the 3’ untranslated region (UTR) of the <em>msl-2</em> transcript and represses translation initiation. The mechanism exerted by Hrp48 during translation repression and its interaction with <em>msl-2</em> are not well understood. Here we investigate the RNA binding specificity and affinity of the tandem RNA recognition motifs of Hrp48. Using NMR spectroscopy, molecular dynamics simulations and isothermal titration calorimetry, we identified the exact region of <em>msl-2</em> 3’ UTR recognized by Hrp48. Additional biophysical experiments and translation assays give further insights into complex formation of Hrp48, Unr, Sxl and RNA. Our results show that Hrp48 binds independent of Sxl and Unr downstream of the E and F binding sites of Sxl and Unr to <em>msl-2</em>.</div></div>","PeriodicalId":8979,"journal":{"name":"Biophysical chemistry","volume":null,"pages":null},"PeriodicalIF":3.3,"publicationDate":"2024-10-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142590115","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Understanding Cu+2 binding with DNA: A molecular dynamics study comparing Cu2+ and Mg2+ binding to the Dickerson DNA 了解 Cu+2 与 DNA 的结合:比较 Cu2+ 和 Mg2+ 与 Dickerson DNA 结合的分子动力学研究。
IF 3.3 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-10-28 DOI: 10.1016/j.bpc.2024.107347
Cu2+ ions led DNA damage by reactive oxygen species (ROS) is widely known biological phenomena. The ionic radii of Cu2+ and Mg2+ being similar, the binding of Cu2+ ions to DNA is expected to be similar to that of the Mg2+ ions. However, little is known how Cu2+ ions bind in different parts (phosphate, major and minor grooves) of a double-strand (ds) DNA, especially at atomic level. In the present study, we employ molecular dynamic (MD) simulations to investigate the binding of Cu2+ ions with the Dickerson DNA, a B-type dodecamer double stranded (ds) DNA. The binding characteristics of Cu2+ and Mg2+ ions with this dsDNA are compared to get an insight into the differences and similarities in binding behavior of both ions. Unlike Mg2+ ions, the first hydration shell of Cu2+ is found to be labile, thus it shows both direct and indirect binding with the dsDNA, i.e., binding through displacement of water from the hydration shell or through the hydration shell. Though the binding propensity of Cu2+ ions with dsDNA is observed relatively stronger, the binding order to phosphates, major groove, and minor groove is found qualitatively similar (phosphates > major groove > minor groove) for both ions. The study gives a deep understanding of Cu2+ binding to DNA, which could be helpful in rationalizing the Cu2+ led ROS-mediated DNA damage.
Cu2+ 离子导致活性氧(ROS)损伤 DNA 是广为人知的生物现象。Cu2+ 和 Mg2+ 的离子半径相似,因此 Cu2+ 离子与 DNA 的结合情况预计与 Mg2+ 离子相似。然而,人们对 Cu2+ 离子如何与双链(ds)DNA 的不同部分(磷酸、主要沟槽和次要沟槽)结合,尤其是在原子水平上结合知之甚少。在本研究中,我们采用分子动力学(MD)模拟来研究 Cu2+ 离子与 Dickerson DNA(一种 B 型十二聚体双链(ds)DNA)的结合。通过比较 Cu2+ 离子和 Mg2+ 离子与该 dsDNA 的结合特性,可以深入了解两种离子结合行为的异同。与 Mg2+ 离子不同,Cu2+ 离子的第一个水合壳是易变的,因此它与 dsDNA 的结合既有直接结合,也有间接结合,即通过水合壳中的水置换结合或通过水合壳结合。虽然观察到 Cu2+ 离子与 dsDNA 的结合倾向相对较强,但发现这两种离子与磷酸盐、主沟和小沟的结合顺序在性质上相似(磷酸盐 > 主沟 > 小沟)。该研究深入揭示了 Cu2+ 与 DNA 的结合,有助于合理解释 Cu2+ 导致的 ROS 介导的 DNA 损伤。
{"title":"Understanding Cu+2 binding with DNA: A molecular dynamics study comparing Cu2+ and Mg2+ binding to the Dickerson DNA","authors":"","doi":"10.1016/j.bpc.2024.107347","DOIUrl":"10.1016/j.bpc.2024.107347","url":null,"abstract":"<div><div>Cu<sup>2+</sup> ions led DNA damage by reactive oxygen species (ROS) is widely known biological phenomena. The ionic radii of Cu<sup>2+</sup> and Mg<sup>2+</sup> being similar, the binding of Cu<sup>2+</sup> ions to DNA is expected to be similar to that of the Mg<sup>2+</sup> ions. However, little is known how Cu<sup>2+</sup> ions bind in different parts (phosphate, major and minor grooves) of a double-strand (ds) DNA, especially at atomic level. In the present study, we employ molecular dynamic (MD) simulations to investigate the binding of Cu<sup>2+</sup> ions with the Dickerson DNA, a B-type dodecamer double stranded (ds) DNA. The binding characteristics of Cu<sup>2+</sup> and Mg<sup>2+</sup> ions with this dsDNA are compared to get an insight into the differences and similarities in binding behavior of both ions. Unlike Mg<sup>2+</sup> ions, the first hydration shell of Cu<sup>2+</sup> is found to be labile, thus it shows both direct and indirect binding with the dsDNA, i.e., binding through displacement of water from the hydration shell or through the hydration shell. Though the binding propensity of Cu<sup>2+</sup> ions with dsDNA is observed relatively stronger, the binding order to phosphates, major groove, and minor groove is found qualitatively similar (phosphates &gt; major groove &gt; minor groove) for both ions. The study gives a deep understanding of Cu<sup>2+</sup> binding to DNA, which could be helpful in rationalizing the Cu<sup>2+</sup> led ROS-mediated DNA damage.</div></div>","PeriodicalId":8979,"journal":{"name":"Biophysical chemistry","volume":null,"pages":null},"PeriodicalIF":3.3,"publicationDate":"2024-10-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142567741","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Biophysical significance of fluorescence spectroscopy in deciphering nucleic acid dynamics: From fundamental to recent advancements 荧光光谱在解读核酸动力学方面的生物物理意义:从基础到最新进展
IF 3.3 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-10-22 DOI: 10.1016/j.bpc.2024.107345
Fluorescence spectroscopy has revolutionized the study of nucleic acids, providing invaluable insights into the dynamic processes that underpin gene expression, replication, and repair. This review explores the application of fluorescence probes in monitoring the conformational changes, interactions, and regulatory mechanisms of DNA and RNA. We discuss the utility of intercalating and non-intercalating fluorescent probes in real-time tracking of nucleic acid dynamics, highlighting their role in elucidating the molecular mechanisms of DNA replication, transcriptional regulation, and DNA repair. By offering a detailed analysis of recent advancements, this review underscores the significance of fluorescence-based techniques in enhancing our understanding of nucleic acid behavior and their implications for genomic stability and therapeutic interventions.
荧光光谱学彻底改变了核酸研究,为了解基因表达、复制和修复的动态过程提供了宝贵的见解。本综述探讨了荧光探针在监测 DNA 和 RNA 的构象变化、相互作用和调控机制方面的应用。我们讨论了插层和非插层荧光探针在实时跟踪核酸动态中的应用,强调了它们在阐明 DNA 复制、转录调控和 DNA 修复的分子机制中的作用。通过对最新进展的详细分析,这篇综述强调了基于荧光的技术在加深我们对核酸行为及其对基因组稳定性和治疗干预的影响的理解方面所具有的重要意义。
{"title":"Biophysical significance of fluorescence spectroscopy in deciphering nucleic acid dynamics: From fundamental to recent advancements","authors":"","doi":"10.1016/j.bpc.2024.107345","DOIUrl":"10.1016/j.bpc.2024.107345","url":null,"abstract":"<div><div>Fluorescence spectroscopy has revolutionized the study of nucleic acids, providing invaluable insights into the dynamic processes that underpin gene expression, replication, and repair. This review explores the application of fluorescence probes in monitoring the conformational changes, interactions, and regulatory mechanisms of DNA and RNA. We discuss the utility of intercalating and non-intercalating fluorescent probes in real-time tracking of nucleic acid dynamics, highlighting their role in elucidating the molecular mechanisms of DNA replication, transcriptional regulation, and DNA repair. By offering a detailed analysis of recent advancements, this review underscores the significance of fluorescence-based techniques in enhancing our understanding of nucleic acid behavior and their implications for genomic stability and therapeutic interventions.</div></div>","PeriodicalId":8979,"journal":{"name":"Biophysical chemistry","volume":null,"pages":null},"PeriodicalIF":3.3,"publicationDate":"2024-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142534624","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
How fingers affect folding of a thumb: Inter-subdomain cooperation in the folding of SARS-CoV-2 RdRp protein 手指如何影响拇指的折叠:SARS-CoV-2 RdRp 蛋白折叠过程中的亚域间合作
IF 3.3 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-10-18 DOI: 10.1016/j.bpc.2024.107342
The RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 is a critical enzyme essential for the virus's replication and transcription, making it a key therapeutic target. The RdRp protein exhibits a characteristic cupped right-hand shaped structure with two vital subdomains: the fingers and the thumb. Despite being distinct, biophysical experiments suggest that these subdomains cooperate to facilitate RNA accommodation, ensuring RdRp functionality. To investigate the structure-based mechanisms underlying the fingers-thumb interaction in both apo and RNA-bound RdRp, we constructed a coarse-grained structure-based model based on recent cryo-electron microscopy data. The simulations reveal frequent open-to-closed conformational transitions in apo RdRp, akin to a breathing-like motion. These conformational changes are regulated by the fingers-thumb association and the folding dynamics of the thumb subdomain. The thumb adopts a stable fold only when tethered by the fingers-thumb interface; when these subdomains are disconnected, the thumb transitions into an open state. A significant number of open-to-closed transition events were analyzed to generate a transition contact probability map, which highlights a few specific residues at the thumb-fingers interface, distant from the RNA accommodation sites, as essential for inducing the thumb's folding process. Given that thumb subdomain folding is critical for RNA binding and viral replication, the study proposes that these interfacial residues may function as remote regulatory switches and could be targeted for the development of allosteric drugs against SARS-CoV-2 and similar RNA viruses.
SARS-CoV-2 的 RNA 依赖性 RNA 聚合酶(RdRp)是病毒复制和转录所必需的关键酶,因此成为关键的治疗靶点。RdRp 蛋白具有独特的右手杯状结构,有两个重要的子域:手指和拇指。尽管各不相同,但生物物理实验表明,这两个亚域相互合作,促进 RNA 的容纳,确保了 RdRp 的功能。为了探究apo和RNA结合的RdRp中手指与拇指相互作用的结构机制,我们根据最近的冷冻电镜数据构建了一个粗粒度的结构模型。模拟结果表明,在 apo RdRp 中,从打开到关闭的构象转变非常频繁,类似于呼吸运动。这些构象变化受手指与拇指的关联以及拇指亚域的折叠动力学的调控。拇指只有在被手指-拇指界面拴住时才会采用稳定的折叠;当这些子域断开时,拇指就会过渡到开放状态。通过分析大量从打开到关闭的转换事件,生成了转换接触概率图,该图突出显示了拇指-手指界面上远离 RNA 容纳位点的几个特定残基,它们对于诱导拇指的折叠过程至关重要。鉴于拇指亚域的折叠对 RNA 结合和病毒复制至关重要,该研究认为这些界面残基可能具有远程调控开关的功能,可以作为开发针对 SARS-CoV-2 和类似 RNA 病毒的异构药物的目标。
{"title":"How fingers affect folding of a thumb: Inter-subdomain cooperation in the folding of SARS-CoV-2 RdRp protein","authors":"","doi":"10.1016/j.bpc.2024.107342","DOIUrl":"10.1016/j.bpc.2024.107342","url":null,"abstract":"<div><div>The RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 is a critical enzyme essential for the virus's replication and transcription, making it a key therapeutic target. The RdRp protein exhibits a characteristic cupped right-hand shaped structure with two vital subdomains: the fingers and the thumb. Despite being distinct, biophysical experiments suggest that these subdomains cooperate to facilitate RNA accommodation, ensuring RdRp functionality. To investigate the structure-based mechanisms underlying the fingers-thumb interaction in both apo and RNA-bound RdRp, we constructed a coarse-grained structure-based model based on recent cryo-electron microscopy data. The simulations reveal frequent open-to-closed conformational transitions in apo RdRp, akin to a breathing-like motion. These conformational changes are regulated by the fingers-thumb association and the folding dynamics of the thumb subdomain. The thumb adopts a stable fold only when tethered by the fingers-thumb interface; when these subdomains are disconnected, the thumb transitions into an open state. A significant number of open-to-closed transition events were analyzed to generate a transition contact probability map, which highlights a few specific residues at the thumb-fingers interface, distant from the RNA accommodation sites, as essential for inducing the thumb's folding process. Given that thumb subdomain folding is critical for RNA binding and viral replication, the study proposes that these interfacial residues may function as remote regulatory switches and could be targeted for the development of allosteric drugs against SARS-CoV-2 and similar RNA viruses.</div></div>","PeriodicalId":8979,"journal":{"name":"Biophysical chemistry","volume":null,"pages":null},"PeriodicalIF":3.3,"publicationDate":"2024-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142534111","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
In vitro and in silico effect of meldrum's acid-derived compounds on Staphylococcus aureus strains as NorA efflux pump inhibitors 作为 NorA 外排泵抑制剂,梅尔德隆酸衍生化合物对金黄色葡萄球菌菌株的体外和硅学效应。
IF 3.3 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-10-18 DOI: 10.1016/j.bpc.2024.107344
The misuse of antibiotics has led to an alarming increase in bacterial strains resistant to these drugs. Efflux pumps, which expel antibiotics from bacterial cells, have emerged as one of the key mechanisms of bacterial resistance. In the quest to combat and mitigate bacterial resistance, researchers have turned their attention to efflux pump inhibitors as a potential solution. Meldrum's acid, a synthetic molecule widely utilized in the synthesis of bioactive compounds, has garnered significant interest in this regard. Hence, this study aims to investigate the antibacterial activity and evaluate the efficacy of three derivatives of meldrum's acid in inhibiting efflux mechanisms, employing both in silico and in vitro approaches. The antibacterial activity of the derivatives was assessed through rigorous broth microdilution testing. While the derivatives themselves did not exhibit direct antibacterial activity, they demonstrated remarkable potential in potentiating the effects of antibiotics. Additionally, fluorescence emission assays using ethidium bromide (EtBr) revealed fluorescence levels comparable to the positive control, indicating a possible blockade of efflux pumps. Molecular docking studies conducted in silico further supported these findings by revealing binding interactions similar to norfloxacin and CCCP, known efflux pump inhibitors. These results underscore the potential of meldrum's acid derivatives as effective inhibitors of efflux pumps. By inhibiting these mechanisms, the derivatives hold promise in enhancing the effectiveness of antibiotics and combatting bacterial resistance. This study contributes valuable insights into the development of novel strategies to address the pressing issue of bacterial resistance and paves the way for further research and exploration in this field.
抗生素的滥用导致对这些药物产生抗药性的细菌菌株数量惊人地增加。将抗生素排出细菌细胞的外排泵已成为细菌产生耐药性的关键机制之一。为了消除和减轻细菌的耐药性,研究人员将目光转向了外排泵抑制剂,将其作为一种潜在的解决方案。梅尔杜姆酸是一种广泛用于合成生物活性化合物的合成分子,在这方面引起了极大的兴趣。因此,本研究采用了硅学和体外方法,旨在研究美杜莎酸的三种衍生物的抗菌活性,并评估其在抑制外流机制方面的功效。衍生物的抗菌活性是通过严格的肉汤微稀释试验进行评估的。虽然这些衍生物本身没有表现出直接的抗菌活性,但它们在增强抗生素效果方面表现出了显著的潜力。此外,使用溴化乙锭(EtBr)进行的荧光发射测定显示,其荧光水平与阳性对照相当,这表明它们可能阻断了外排泵。在硅学中进行的分子对接研究进一步证实了这些发现,研究显示了与诺氟沙星和CCCP(已知的外排泵抑制剂)类似的结合相互作用。这些结果凸显了美杜莎酸衍生物作为有效外排泵抑制剂的潜力。通过抑制这些机制,这些衍生物有望提高抗生素的疗效并对抗细菌耐药性。这项研究为开发解决细菌耐药性这一紧迫问题的新策略提供了宝贵的见解,并为这一领域的进一步研究和探索铺平了道路。
{"title":"In vitro and in silico effect of meldrum's acid-derived compounds on Staphylococcus aureus strains as NorA efflux pump inhibitors","authors":"","doi":"10.1016/j.bpc.2024.107344","DOIUrl":"10.1016/j.bpc.2024.107344","url":null,"abstract":"<div><div>The misuse of antibiotics has led to an alarming increase in bacterial strains resistant to these drugs. Efflux pumps, which expel antibiotics from bacterial cells, have emerged as one of the key mechanisms of bacterial resistance. In the quest to combat and mitigate bacterial resistance, researchers have turned their attention to efflux pump inhibitors as a potential solution. Meldrum's acid, a synthetic molecule widely utilized in the synthesis of bioactive compounds, has garnered significant interest in this regard. Hence, this study aims to investigate the antibacterial activity and evaluate the efficacy of three derivatives of meldrum's acid in inhibiting efflux mechanisms, employing both in silico and in vitro approaches. The antibacterial activity of the derivatives was assessed through rigorous broth microdilution testing. While the derivatives themselves did not exhibit direct antibacterial activity, they demonstrated remarkable potential in potentiating the effects of antibiotics. Additionally, fluorescence emission assays using ethidium bromide (EtBr) revealed fluorescence levels comparable to the positive control, indicating a possible blockade of efflux pumps. Molecular docking studies conducted in silico further supported these findings by revealing binding interactions similar to norfloxacin and CCCP, known efflux pump inhibitors. These results underscore the potential of meldrum's acid derivatives as effective inhibitors of efflux pumps. By inhibiting these mechanisms, the derivatives hold promise in enhancing the effectiveness of antibiotics and combatting bacterial resistance. This study contributes valuable insights into the development of novel strategies to address the pressing issue of bacterial resistance and paves the way for further research and exploration in this field.</div></div>","PeriodicalId":8979,"journal":{"name":"Biophysical chemistry","volume":null,"pages":null},"PeriodicalIF":3.3,"publicationDate":"2024-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142494593","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Solubilisation & purification of membrane proteins using benzylamine-modified SMA polymers 使用苄胺改性 SMA 聚合物增溶和纯化膜蛋白。
IF 3.3 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-10-18 DOI: 10.1016/j.bpc.2024.107343
Extraction of proteins from the membrane using styrene maleic acid co-polymers (SMA), forming SMA lipid particles (SMALPs), has allowed for the first time the purification of membrane proteins with their lipid bilayer environment. To date, SMA2000 has been the most effective polymer used for this purpose, with a 2:1 ratio of styrene:maleic acid, and styrene and maleic acid moieties spread statistically throughout the chain. However, SMA2000 is a highly polydisperse polymer that contains an array of different polymer lengths and sequences. RAFT polymerisation offers much better control over the polymer length; however, homogeneous distribution of styrene and maleic acid throughout the polymer is difficult to achieve. Instead, here RAFT polymerisation was used to produce a 1:1 styrene:maleic anhydride polymer, which was then modified with benzylamine. This mimics the 2:1 hydrophobic:hydrophilic nature of SMA2000, while controlling the length and obtaining a homogeneous distribution of the hydrophobic moieties (styrene and N-benzylmaleimide). SMA-benzylamine (SMA-BA) polymers of three different lengths (2, 4, and 7 kDa) were all able to solubilise purified lipids, cellular membranes, and a range of specific proteins. However, the larger 7 kDa polymer solubilised membranes more slowly and less efficiently than the shorter polymers. This also affected the yield of purified protein obtained by affinity purification with this polymer. The smallest 2 kDa polymer solubilised membranes the fastest but appeared to offer less stability to the extracted proteins. The SMA-BA polymers were more sensitive to Mg2+ ions than SMA2000. SMA-BA 4 kDa was otherwise comparable to SMA2000 and even gave a higher degree of purity.
使用苯乙烯-马来酸共聚物(SMA)从膜中提取蛋白质,形成 SMA 脂质颗粒(SMALPs),首次实现了在脂质双分子层环境中纯化膜蛋白质。迄今为止,SMA2000 是用于此目的最有效的聚合物,其苯乙烯和马来酸的比例为 2:1,苯乙烯和马来酸分子分布在整个链中。不过,SMA2000 是一种高度分散的聚合物,含有一系列不同长度和序列的聚合物。RAFT 聚合可以更好地控制聚合物的长度;但是,很难实现苯乙烯和马来酸在整个聚合物中的均匀分布。因此,这里采用了 RAFT 聚合法来生产 1:1 苯乙烯:马来酸酐聚合物,然后用苄胺对其进行改性。这就模仿了 SMA2000 2:1 的疏水:亲水性质,同时控制了长度并获得了疏水分子(苯乙烯和 N-苄基马来酰亚胺)的均匀分布。三种不同长度(2、4 和 7 kDa)的 SMA-苄胺(SMA-BA)聚合物都能溶解纯化的脂质、细胞膜和一系列特定蛋白质。不过,与较短的聚合物相比,较大的 7 kDa 聚合物溶解膜的速度更慢,效率更低。这也影响了使用这种聚合物进行亲和纯化所获得的纯化蛋白质的产量。最小的 2 kDa 聚合物溶解膜的速度最快,但提取蛋白质的稳定性似乎较差。与 SMA2000 相比,SMA-BA 聚合物对 Mg2+ 离子更敏感。SMA-BA 4 kDa 在其他方面与 SMA2000 相当,甚至纯度更高。
{"title":"Solubilisation & purification of membrane proteins using benzylamine-modified SMA polymers","authors":"","doi":"10.1016/j.bpc.2024.107343","DOIUrl":"10.1016/j.bpc.2024.107343","url":null,"abstract":"<div><div>Extraction of proteins from the membrane using styrene maleic acid <em>co</em>-polymers (SMA), forming SMA lipid particles (SMALPs), has allowed for the first time the purification of membrane proteins with their lipid bilayer environment. To date, SMA2000 has been the most effective polymer used for this purpose, with a 2:1 ratio of styrene:maleic acid, and styrene and maleic acid moieties spread statistically throughout the chain. However, SMA2000 is a highly polydisperse polymer that contains an array of different polymer lengths and sequences. RAFT polymerisation offers much better control over the polymer length; however, homogeneous distribution of styrene and maleic acid throughout the polymer is difficult to achieve. Instead, here RAFT polymerisation was used to produce a 1:1 styrene:maleic anhydride polymer, which was then modified with benzylamine. This mimics the 2:1 hydrophobic:hydrophilic nature of SMA2000, while controlling the length and obtaining a homogeneous distribution of the hydrophobic moieties (styrene and <em>N</em>-benzylmaleimide). SMA-benzylamine (SMA-BA) polymers of three different lengths (2, 4, and 7 kDa) were all able to solubilise purified lipids, cellular membranes, and a range of specific proteins. However, the larger 7 kDa polymer solubilised membranes more slowly and less efficiently than the shorter polymers. This also affected the yield of purified protein obtained by affinity purification with this polymer. The smallest 2 kDa polymer solubilised membranes the fastest but appeared to offer less stability to the extracted proteins. The SMA-BA polymers were more sensitive to Mg<sup>2+</sup> ions than SMA2000. SMA-BA 4 kDa was otherwise comparable to SMA2000 and even gave a higher degree of purity.</div></div>","PeriodicalId":8979,"journal":{"name":"Biophysical chemistry","volume":null,"pages":null},"PeriodicalIF":3.3,"publicationDate":"2024-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142494594","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
On the production of singlet oxygen by the isoalloxazine ring in free and protein-bound flavin cofactors 异咯嗪环在自由和蛋白质结合的黄素辅因子中产生单线态氧
IF 3.3 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-10-10 DOI: 10.1016/j.bpc.2024.107333
Flavin cofactors, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), as a part of flavoenzymes play a critical role in the catalysis of multiple reactions predominantly of a redox nature. Question arises why nature developed two very similar cofactors with an identical functional part – isoalloxazine ring. We believe that an answer is related to the fact that the isoalloxazine ring belongs to endogenous photosensitizers able to produce reactive and potentially harmful singlet oxygen, 1O2, with high efficiency, ΦΔ,FMN ∼ 0.6. In fact, in contrast with one main conformation of FMN in water, the presence of the adenosine mononucleotide in FAD induces a dynamic equilibrium of two main conformations – closed (∼80 %) and open (∼20 %). The presence of predominant closed conformation of FAD in water has a significant impact on the ΦΔ,FAD value, which is nearly 10-fold lower, ΦΔ,FAD ∼ 0.07, than that of FMN. On the other hand, based on our analysis of a non-homologous dataset of FAD containing 105 proteins, ∼75 % enzyme-bound FAD exists predominantly in open conformations but the ΦΔ values are significantly decreased, ΦΔ < 0.03. We addressed these contradictory observations by analysis of: (i) dependence of ΦΔ,FAD value on opening the FAD conformation by urea and (ii) amino acid propensities for isoalloxazine binding site. We demonstrated that urea-induced destabilization, in 7 M vs 0 M urea, of the closed FAD conformation leads to a ∼ 3-fold increase of ΦΔ, proving the causative relation between ΦΔ value and the flavin cofactor conformation. Detailed examination of the flavoproteins dataset clearly indicated positive propensities of three amino acids: glycine, cysteine, and tryptophan for isoalloxazine ring binding site. We hypothesize that both the closed conformation of free FAD and the arrangement of the isoalloxazine binding site is important for prevention of potentially harmful 1O2 production in cells.
黄素辅助因子,即黄素腺嘌呤二核苷酸(FAD)和黄素单核苷酸(FMN),作为黄酶类的一部分,在催化以氧化还原为主的多种反应中发挥着至关重要的作用。人们不禁要问,为什么自然界会产生两种非常相似的辅助因子,而且其功能部分--异咯嗪环--完全相同。我们认为,答案与以下事实有关:异氮杂环属于内源光敏剂,能够高效地产生具有活性和潜在危害的单线态氧 1O2(ΦΔ,FMN∼ 0.6)。事实上,与 FMN 在水中的一种主要构象不同,FAD 中腺苷单核苷酸的存在导致了两种主要构象的动态平衡--封闭构象(∼80%)和开放构象(∼20%)。FAD 在水中的主要封闭构象对ΦΔ,FAD 值有显著影响,ΦΔ,FAD ∼ 0.07,比 FMN 低近 10 倍。另一方面,根据我们对含有 FAD 的 105 个蛋白质的非同源数据集的分析,75% 的酶结合 FAD 主要以开放构象存在,但其 ΦΔ 值显著降低,Φ Δ < 0.03。针对这些相互矛盾的观察结果,我们进行了分析:(i) 脲打开 FAD 构象对ΦΔ,FAD 值的依赖性;(ii) 异丙嗪结合位点的氨基酸倾向性。我们证明了在 7 M 与 0 M 尿素中,脲诱导的封闭 FAD 构象的不稳定性导致ΦΔ增加了 ∼ 3 倍,证明了ΦΔ值与黄素辅助因子构象之间的因果关系。对黄素蛋白数据集的详细研究清楚地表明,甘氨酸、半胱氨酸和色氨酸这三种氨基酸对于异咯嗪环结合位点具有积极的倾向性。我们推测,游离 FAD 的封闭构象和异丙嗪环结合位点的排列对于防止细胞中产生潜在有害的 1O2 非常重要。
{"title":"On the production of singlet oxygen by the isoalloxazine ring in free and protein-bound flavin cofactors","authors":"","doi":"10.1016/j.bpc.2024.107333","DOIUrl":"10.1016/j.bpc.2024.107333","url":null,"abstract":"<div><div>Flavin cofactors, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), as a part of flavoenzymes play a critical role in the catalysis of multiple reactions predominantly of a redox nature. Question arises why nature developed two very similar cofactors with an identical functional part – isoalloxazine ring. We believe that an answer is related to the fact that the isoalloxazine ring belongs to endogenous photosensitizers able to produce reactive and potentially harmful singlet oxygen, <sup>1</sup>O<sub>2</sub>, with high efficiency, Φ<sub>Δ,FMN</sub> ∼ 0.6. In fact, in contrast with one main conformation of FMN in water, the presence of the adenosine mononucleotide in FAD induces a dynamic equilibrium of two main conformations – closed (∼80 %) and open (∼20 %). The presence of predominant closed conformation of FAD in water has a significant impact on the Φ<sub>Δ,FAD</sub> value, which is nearly 10-fold lower, Φ<sub>Δ,FAD</sub> ∼ 0.07, than that of FMN. On the other hand, based on our analysis of a non-homologous dataset of FAD containing 105 proteins, ∼75 % enzyme-bound FAD exists predominantly in open conformations but the Φ<sub>Δ</sub> values are significantly decreased, Φ<sub>Δ</sub> &lt; 0.03. We addressed these contradictory observations by analysis of: (i) dependence of Φ<sub>Δ,FAD</sub> value on opening the FAD conformation by urea and (ii) amino acid propensities for isoalloxazine binding site. We demonstrated that urea-induced destabilization, in 7 M vs 0 M urea, of the closed FAD conformation leads to a ∼ 3-fold increase of Φ<sub>Δ</sub>, proving the causative relation between Φ<sub>Δ</sub> value and the flavin cofactor conformation. Detailed examination of the flavoproteins dataset clearly indicated positive propensities of three amino acids: glycine, cysteine, and tryptophan for isoalloxazine ring binding site. We hypothesize that both the closed conformation of free FAD and the arrangement of the isoalloxazine binding site is important for prevention of potentially harmful <sup>1</sup>O<sub>2</sub> production in cells.</div></div>","PeriodicalId":8979,"journal":{"name":"Biophysical chemistry","volume":null,"pages":null},"PeriodicalIF":3.3,"publicationDate":"2024-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142436535","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Stability and conformation of DNA-hairpin in cylindrical confinement DNA 发夹在圆柱约束下的稳定性和构象。
IF 3.3 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-09-30 DOI: 10.1016/j.bpc.2024.107331
We conducted atomistic Molecular Dynamics (MD) simulations of DNA-Hairpin molecules encapsulated within Single-Walled Carbon Nanotubes (SWCNTs) at a temperature of 300 K. Our investigation revealed that the structural integrity of the DNA-Hairpin can be maintained within SWCNTs, provided that the diameter of the SWCNT exceeds a critical threshold value. Conversely, when the SWCNT diameter falls below this critical threshold, the DNA-Hairpin undergoes denaturation, even at a temperature of 300 K. The DNA-Hairpin model we employed consisted of a 12-base pair stem and a 3-base loop, and we studied various SWCNTs with different diameters. Our analyses identified a critical SWCNT diameter of 3.39 nm at 300 K. Examination of key structural features, such as hydrogen bonds (H-bonds), van der Waals (vdW) interactions, and other inter-base interactions, demonstrated a significant reduction in the number of H-bonds, vdW energy, and electrostatic energies among the DNA hairpin's constituent bases when confined within narrower SWCNTs (with diameters of 2.84 nm and 3.25 nm). However, it was observed that the increased interaction energy between the DNA-Hairpin and the inner surface of narrower SWCNTs promoted the denaturation of the DNA-Hairpin. In-depth analysis of electrostatic mapping and hydration status further revealed that the DNA-Hairpin experienced inadequate hydration and non-uniform distribution of counter ions within SWCNTs having diameters below the critical value of 3.39 nm. Our inference is that the inappropriate hydration of counter ions, along with their non-uniform spatial distribution around the DNA hairpin, contributes to the denaturation of the molecule within SWCNTs of smaller diameters. For DNA-Hairpin molecules that remained undenatured within SWCNTs, we investigated their mechanical properties, particularly the elastic properties. Our findings demonstrated an increase in the persistence length of the DNA-Hairpin with increasing SWCNT diameter. Additionally, the stretch modulus and torsional stiffness of the DNA-Hairpin were observed to increase as a function of SWCNT diameter, indicating that confinement within SWCNTs enhances the mechanical flexibility of the DNA-Hairpin.
我们对在 300 K 温度下封装在单壁碳纳米管(SWCNT)中的 DNA-Hairpin 分子进行了原子分子动力学(MD)模拟。我们的研究发现,只要单壁碳纳米管的直径超过临界阈值,DNA-Hairpin 的结构完整性就能在单壁碳纳米管中保持不变。相反,当 SWCNT 直径低于临界阈值时,DNA-Hairpin 会发生变性,即使在 300 K 的温度下也是如此。我们采用的 DNA-Hairpin 模型包括一个 12 碱基对的茎和一个 3 碱基环,我们研究了不同直径的 SWCNT。对氢键 (H-bonds)、范德华 (vdW) 相互作用和其他碱基间相互作用等关键结构特征的研究表明,当被限制在较窄的 SWCNT(直径为 2.84 nm 和 3.25 nm)中时,DNA 发夹的组成碱基之间的 H 键数量、vdW 能量和静电能量显著减少。不过,据观察,DNA 发夹与较窄的 SWCNT 内表面之间的相互作用能增加,促进了 DNA 发夹的变性。对静电映射和水合状态的深入分析进一步表明,DNA-Hairpin 在直径低于临界值 3.39 nm 的 SWCNT 中水合不充分,反离子分布不均匀。我们的推断是,反离子的不适当水合及其在 DNA 发夹周围的不均匀空间分布导致了分子在直径较小的 SWCNT 内变性。对于在 SWCNT 内保持未变性的 DNA 发夹蛋白分子,我们研究了它们的机械特性,尤其是弹性特性。我们的研究结果表明,DNA-Hairpin 的持续长度随着 SWCNT 直径的增加而增加。此外,我们还观察到 DNA-Hairpin 的拉伸模量和扭转刚度随 SWCNT 直径的增加而增加,这表明在 SWCNT 内的限制增强了 DNA-Hairpin 的机械灵活性。
{"title":"Stability and conformation of DNA-hairpin in cylindrical confinement","authors":"","doi":"10.1016/j.bpc.2024.107331","DOIUrl":"10.1016/j.bpc.2024.107331","url":null,"abstract":"<div><div>We conducted atomistic Molecular Dynamics (MD) simulations of DNA-Hairpin molecules encapsulated within Single-Walled Carbon Nanotubes (SWCNTs) at a temperature of 300 K. Our investigation revealed that the structural integrity of the DNA-Hairpin can be maintained within SWCNTs, provided that the diameter of the SWCNT exceeds a critical threshold value. Conversely, when the SWCNT diameter falls below this critical threshold, the DNA-Hairpin undergoes denaturation, even at a temperature of 300 K. The DNA-Hairpin model we employed consisted of a 12-base pair stem and a 3-base loop, and we studied various SWCNTs with different diameters. Our analyses identified a critical SWCNT diameter of 3.39 nm at 300 K. Examination of key structural features, such as hydrogen bonds (H-bonds), van der Waals (vdW) interactions, and other inter-base interactions, demonstrated a significant reduction in the number of H-bonds, vdW energy, and electrostatic energies among the DNA hairpin's constituent bases when confined within narrower SWCNTs (with diameters of 2.84 nm and 3.25 nm). However, it was observed that the increased interaction energy between the DNA-Hairpin and the inner surface of narrower SWCNTs promoted the denaturation of the DNA-Hairpin. In-depth analysis of electrostatic mapping and hydration status further revealed that the DNA-Hairpin experienced inadequate hydration and non-uniform distribution of counter ions within SWCNTs having diameters below the critical value of 3.39 nm. Our inference is that the inappropriate hydration of counter ions, along with their non-uniform spatial distribution around the DNA hairpin, contributes to the denaturation of the molecule within SWCNTs of smaller diameters. For DNA-Hairpin molecules that remained undenatured within SWCNTs, we investigated their mechanical properties, particularly the elastic properties. Our findings demonstrated an increase in the persistence length of the DNA-Hairpin with increasing SWCNT diameter. Additionally, the stretch modulus and torsional stiffness of the DNA-Hairpin were observed to increase as a function of SWCNT diameter, indicating that confinement within SWCNTs enhances the mechanical flexibility of the DNA-Hairpin.</div></div>","PeriodicalId":8979,"journal":{"name":"Biophysical chemistry","volume":null,"pages":null},"PeriodicalIF":3.3,"publicationDate":"2024-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142457279","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Characterization of PARP1 binding to c-KIT1 G-quadruplex DNA: Insights into domain-specific interactions PARP1 与 c-KIT1 G-quadruplex DNA 结合的特征:洞察特定结构域的相互作用。
IF 3.3 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-09-25 DOI: 10.1016/j.bpc.2024.107330
Poly(ADP-ribose) polymerase 1 (PARP1) is a nuclear enzyme involved in catalyzing Poly-(ADP-ribosyl)ation. PARP1 binds to different forms of DNA and DNA breaks and thus plays important roles in several cellular processes, including DNA damage repair, cell cycle regulation, chromatin remodeling, and maintaining genomic stability. In this study, we conducted biochemical and biophysical characterization of PARP1 binding to G-quadruplex DNA (G4-DNA). Our investigation identified ZnF1, ZnF3, and WGR as the critical domains to mediate PARP1 binding to G4-c-KIT1. Also, our results show that these domains together show cooperativity for G4-c-KIT1 recognition. Further, we establish that the presence of an oxidized (5-carboxylcytosine) base in the loop region of G4-c-KIT1 (G4-5caC-cKIT1) does not affect its recognition by PARP1. Both G4-c-KIT1 and G4-5caC-cKIT1 are potent stimulators of PARP1's catalytic activity. Our study advances the understanding of PARP1's versatile DNA binding capabilities for G4-c-KIT1 DNA irrespective of the oxidation/ modification in the DNA base. These insights into PARP1's domain-specific contributions to G4-c-KIT1 DNA recognition and catalysis expand our knowledge of its multifaceted roles in DNA repair and genome maintenance.
聚(ADP-核糖)聚合酶 1(PARP1)是一种参与催化聚(ADP-核糖)合成的核酶。PARP1 与不同形式的 DNA 和 DNA 断裂结合,因此在 DNA 损伤修复、细胞周期调控、染色质重塑和维持基因组稳定性等多个细胞过程中发挥着重要作用。在这项研究中,我们对 PARP1 与 G 型四倍体 DNA(G4-DNA)的结合进行了生化和生物物理鉴定。我们的研究发现 ZnF1、ZnF3 和 WGR 是介导 PARP1 与 G4-c-KIT1 结合的关键结构域。同时,我们的研究结果表明,这些结构域在 G4-c-KIT1 识别过程中具有协同作用。此外,我们还证实,G4-c-KIT1(G4-5caC-KIT1)的环状区域中存在氧化(5-羧基胞嘧啶)碱基不会影响 PARP1 对其的识别。G4-c-KIT1 和 G4-5caC-cKIT1 都能有效刺激 PARP1 的催化活性。我们的研究加深了人们对 PARP1 与 G4-c-KIT1 DNA 的多功能 DNA 结合能力的理解,无论 DNA 碱基的氧化/修饰情况如何。这些关于 PARP1 对 G4-c-KIT1 DNA 识别和催化的特异领域贡献的见解,拓展了我们对其在 DNA 修复和基因组维护中的多方面作用的认识。
{"title":"Characterization of PARP1 binding to c-KIT1 G-quadruplex DNA: Insights into domain-specific interactions","authors":"","doi":"10.1016/j.bpc.2024.107330","DOIUrl":"10.1016/j.bpc.2024.107330","url":null,"abstract":"<div><div>Poly(ADP-ribose) polymerase 1 (PARP1) is a nuclear enzyme involved in catalyzing Poly-(ADP-ribosyl)ation. PARP1 binds to different forms of DNA and DNA breaks and thus plays important roles in several cellular processes, including DNA damage repair, cell cycle regulation, chromatin remodeling, and maintaining genomic stability. In this study, we conducted biochemical and biophysical characterization of PARP1 binding to G-quadruplex DNA (G4-DNA). Our investigation identified ZnF1, ZnF3, and WGR as the critical domains to mediate PARP1 binding to G4-c-KIT1. Also, our results show that these domains together show cooperativity for G4-c-KIT1 recognition. Further, we establish that the presence of an oxidized (5-carboxylcytosine) base in the loop region of G4-c-KIT1 (G4-5caC-cKIT1) does not affect its recognition by PARP1. Both G4-c-KIT1 and G4-5caC-cKIT1 are potent stimulators of PARP1's catalytic activity. Our study advances the understanding of PARP1's versatile DNA binding capabilities for G4-c-KIT1 DNA irrespective of the oxidation/ modification in the DNA base. These insights into PARP1's domain-specific contributions to G4-c-KIT1 DNA recognition and catalysis expand our knowledge of its multifaceted roles in DNA repair and genome maintenance.</div></div>","PeriodicalId":8979,"journal":{"name":"Biophysical chemistry","volume":null,"pages":null},"PeriodicalIF":3.3,"publicationDate":"2024-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142341033","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Structural changes of Natronomonas pharaonis halorhodopsin in its late photocycle revealed by solid-state NMR spectroscopy 固态核磁共振光谱揭示 Natronomonas pharaonis halorhodopsin 在光周期后期的结构变化。
IF 3.3 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-09-24 DOI: 10.1016/j.bpc.2024.107329
Natronomonas pharaonis halorhodopsin (NpHR) is a light-driven Cl inward pump that is widely used as an optogenetic tool. Although NpHR is previously extensively studied, its Cl uptake process is not well understood from the protein structure perspective, mainly because in crystalline lattice, it has been difficult to analyze the structural changes associated with the Cl uptake process. In this study, we used solid-state NMR to analyze NpHR both in the Cl-bound and -free states under near-physiological transmembrane condition. Chemical shift perturbation analysis suggested that while the structural change caused by the Cl depletion is widespread over the NpHR molecule, residues in the extracellular (EC) part of helix D exhibited significant conformational changes that may be related to the Cl uptake process. By combining photochemical analysis and dynamic nuclear polarization (DNP)-enhanced solid-state NMR measurement on NpHR point mutants for the suggested residues, we confirmed their importance in the Cl uptake process. In particular, we found the mutation at Ala165 position, located at the trimer interface, to an amino acid with bulky sidechain (A165V) significantly perturbs the late photocycle and disrupts its trimeric assembly in the Cl-free state as well as during the ion-pumping cycle under the photo-irradiated condition. This strongly suggested an outward movement of helix D at EC part, disrupting the trimer integrity. Together with the spectroscopic data and known NpHR crystal structures, we proposed a model that this helix movement is required for creating the Cl entrance path on the extracellular surface of the protein and is crucial to the Cl uptake process.
Natronomonas pharaonis halorhodopsin(NpHR)是一种光驱动的Cl-内向泵,被广泛用作光遗传学工具。虽然此前对 NpHR 进行了广泛的研究,但从蛋白质结构的角度来看,对其 Cl- 摄取过程的了解并不深入,这主要是因为在晶体晶格中,很难分析与 Cl- 摄取过程相关的结构变化。在本研究中,我们利用固态核磁共振分析了 NpHR 在接近生理跨膜条件下的结合态和游离态。化学位移扰动分析表明,Cl-耗竭引起的结构变化遍及整个 NpHR 分子,但螺旋 D 细胞外(EC)部分的残基表现出显著的构象变化,这可能与 Cl-吸收过程有关。通过结合光化学分析和动态核偏振(DNP)增强固态核磁共振测量 NpHR 的点突变体,我们证实了这些残基在 Cl- 摄取过程中的重要性。特别是,我们发现位于三聚体界面的 Ala165 位氨基酸(A165V)突变为一个具有稠厚侧链的氨基酸(A165V)会显著扰乱后期的光周期,破坏其在无 Cl 状态下的三聚体组装,以及在光照射条件下的离子泵循环。这强烈表明,EC 部分的螺旋 D 向外移动,破坏了三聚体的完整性。结合光谱数据和已知的 NpHR 晶体结构,我们提出了一个模型,即这种螺旋运动是在蛋白质胞外表面创建 Cl- 入口通道所必需的,对 Cl- 吸收过程至关重要。
{"title":"Structural changes of Natronomonas pharaonis halorhodopsin in its late photocycle revealed by solid-state NMR spectroscopy","authors":"","doi":"10.1016/j.bpc.2024.107329","DOIUrl":"10.1016/j.bpc.2024.107329","url":null,"abstract":"<div><div><em>Natronomonas pharaonis</em> halorhodopsin (<em>Np</em>HR) is a light-driven Cl<sup>−</sup> inward pump that is widely used as an optogenetic tool. Although <em>Np</em>HR is previously extensively studied, its Cl<sup>−</sup> uptake process is not well understood from the protein structure perspective, mainly because in crystalline lattice, it has been difficult to analyze the structural changes associated with the Cl<sup>−</sup> uptake process. In this study, we used solid-state NMR to analyze <em>Np</em>HR both in the Cl<sup>−</sup>-bound and -free states under near-physiological transmembrane condition. Chemical shift perturbation analysis suggested that while the structural change caused by the Cl<sup>−</sup> depletion is widespread over the <em>Np</em>HR molecule, residues in the extracellular (EC) part of helix D exhibited significant conformational changes that may be related to the Cl<sup>−</sup> uptake process. By combining photochemical analysis and dynamic nuclear polarization (DNP)-enhanced solid-state NMR measurement on <em>Np</em>HR point mutants for the suggested residues, we confirmed their importance in the Cl<sup>−</sup> uptake process. In particular, we found the mutation at Ala165 position, located at the trimer interface, to an amino acid with bulky sidechain (A165V) significantly perturbs the late photocycle and disrupts its trimeric assembly in the Cl<sup>−</sup>-free state as well as during the ion-pumping cycle under the photo-irradiated condition. This strongly suggested an outward movement of helix D at EC part, disrupting the trimer integrity. Together with the spectroscopic data and known <em>Np</em>HR crystal structures, we proposed a model that this helix movement is required for creating the Cl<sup>−</sup> entrance path on the extracellular surface of the protein and is crucial to the Cl<sup>−</sup> uptake process.</div></div>","PeriodicalId":8979,"journal":{"name":"Biophysical chemistry","volume":null,"pages":null},"PeriodicalIF":3.3,"publicationDate":"2024-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142380057","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
期刊
Biophysical chemistry
全部 Acc. Chem. Res. ACS Applied Bio Materials ACS Appl. Electron. Mater. ACS Appl. Energy Mater. ACS Appl. Mater. Interfaces ACS Appl. Nano Mater. ACS Appl. Polym. Mater. ACS BIOMATER-SCI ENG ACS Catal. ACS Cent. Sci. ACS Chem. Biol. ACS Chemical Health & Safety ACS Chem. Neurosci. ACS Comb. Sci. ACS Earth Space Chem. ACS Energy Lett. ACS Infect. Dis. ACS Macro Lett. ACS Mater. Lett. ACS Med. Chem. Lett. ACS Nano ACS Omega ACS Photonics ACS Sens. ACS Sustainable Chem. Eng. ACS Synth. Biol. Anal. Chem. BIOCHEMISTRY-US Bioconjugate Chem. BIOMACROMOLECULES Chem. Res. Toxicol. Chem. Rev. Chem. Mater. CRYST GROWTH DES ENERG FUEL Environ. Sci. Technol. Environ. Sci. Technol. Lett. Eur. J. Inorg. Chem. IND ENG CHEM RES Inorg. Chem. J. Agric. Food. Chem. J. Chem. Eng. Data J. Chem. Educ. J. Chem. Inf. Model. J. Chem. Theory Comput. J. Med. Chem. J. Nat. Prod. J PROTEOME RES J. Am. Chem. Soc. LANGMUIR MACROMOLECULES Mol. Pharmaceutics Nano Lett. Org. Lett. ORG PROCESS RES DEV ORGANOMETALLICS J. Org. Chem. J. Phys. Chem. J. Phys. Chem. A J. Phys. Chem. B J. Phys. Chem. C J. Phys. Chem. Lett. Analyst Anal. Methods Biomater. Sci. Catal. Sci. Technol. Chem. Commun. Chem. Soc. Rev. CHEM EDUC RES PRACT CRYSTENGCOMM Dalton Trans. Energy Environ. Sci. ENVIRON SCI-NANO ENVIRON SCI-PROC IMP ENVIRON SCI-WAT RES Faraday Discuss. Food Funct. Green Chem. Inorg. Chem. Front. Integr. Biol. J. Anal. At. Spectrom. J. Mater. Chem. A J. Mater. Chem. B J. Mater. Chem. C Lab Chip Mater. Chem. Front. Mater. Horiz. MEDCHEMCOMM Metallomics Mol. Biosyst. Mol. Syst. Des. Eng. Nanoscale Nanoscale Horiz. Nat. Prod. Rep. New J. Chem. Org. Biomol. Chem. Org. Chem. Front. PHOTOCH PHOTOBIO SCI PCCP Polym. Chem.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
0
微信
客服QQ
Book学术公众号 扫码关注我们
反馈
×
意见反馈
请填写您的意见或建议
请填写您的手机或邮箱
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
现在去查看 取消
×
提示
确定
Book学术官方微信
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术
文献互助 智能选刊 最新文献 互助须知 联系我们:info@booksci.cn
Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。
Copyright © 2023 Book学术 All rights reserved.
ghs 京公网安备 11010802042870号 京ICP备2023020795号-1