增强脂质:表面活性剂混合胶束中 mTSPO 易位体的结构/功能。

IF 3.3 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY
{"title":"增强脂质:表面活性剂混合胶束中 mTSPO 易位体的结构/功能。","authors":"","doi":"10.1016/j.biochi.2024.04.008","DOIUrl":null,"url":null,"abstract":"<div><p>TSPO is a ubiquitous transmembrane protein used as a pharmacological marker in neuroimaging. The only known atomic structure of mammalian TSPOs comes from the solution NMR of mouse TSPO (mTSPO) bound to the PK11195 ligand and in a DPC surfactant environment. No structure is available in a biomimetic environment and without PK11195 which strongly stiffens the protein.</p><p>We measured the effect of different amphiphilic environments on ligand-free mTSPO to study its structure/function and find optimal solubilization conditions. By replacing the SDS surfactant, where the recombinant protein is purified, with mixed lipid:surfactant (DMPC:DPC) micelles at different ratios (0:1, 1:2, and 2:1, <em>w</em>:<em>w</em>), the α-helix content and interactions and the intrinsic tryptophan (Trp) fluorescence of mTSPO are gradually increased.</p><p>Small-angle X-ray scattering (SAXS) shows a more extended mTSPO/belt complex with the addition of lipids: <em>D</em><sub>max</sub> ∼95 Å in DPC alone <em>versus</em> ∼142 Å in DMPC:DPC (1:2). SEC-MALLS shows that the molecular composition of the mTSPO belt is ∼98 molecules for DPC alone and ∼58 DMPC and ∼175 DPC for DMPC:DPC (1:2). Additionally, DMPC:DPC micelles stabilize mTSPO compared to DPC alone, where the protein has a greater propensity to aggregate. These structural changes are consistent with the increased affinity of mTSPO for the PK11195 ligand in presence of lipids (<em>K</em><sub>d</sub> ∼70 μM in DPC alone <em>versus</em> ∼0.91 μM in DMPC:DPC, 1:2), as measured by microscale thermophoresis (MST).</p><p>In conclusion, mixed lipid:surfactant micelles open new possibilities for the stabilization of membrane proteins and for their study in solution in a more biomimetic amphiphilic environment.</p></div>","PeriodicalId":251,"journal":{"name":"Biochimie","volume":"224 ","pages":"Pages 3-15"},"PeriodicalIF":3.3000,"publicationDate":"2024-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S030090842400083X/pdfft?md5=9e427a6cb180bd19b969cc361f24fb0b&pid=1-s2.0-S030090842400083X-main.pdf","citationCount":"0","resultStr":"{\"title\":\"Enhanced structure/function of mTSPO translocator in lipid:surfactant mixed micelles\",\"authors\":\"\",\"doi\":\"10.1016/j.biochi.2024.04.008\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>TSPO is a ubiquitous transmembrane protein used as a pharmacological marker in neuroimaging. The only known atomic structure of mammalian TSPOs comes from the solution NMR of mouse TSPO (mTSPO) bound to the PK11195 ligand and in a DPC surfactant environment. No structure is available in a biomimetic environment and without PK11195 which strongly stiffens the protein.</p><p>We measured the effect of different amphiphilic environments on ligand-free mTSPO to study its structure/function and find optimal solubilization conditions. By replacing the SDS surfactant, where the recombinant protein is purified, with mixed lipid:surfactant (DMPC:DPC) micelles at different ratios (0:1, 1:2, and 2:1, <em>w</em>:<em>w</em>), the α-helix content and interactions and the intrinsic tryptophan (Trp) fluorescence of mTSPO are gradually increased.</p><p>Small-angle X-ray scattering (SAXS) shows a more extended mTSPO/belt complex with the addition of lipids: <em>D</em><sub>max</sub> ∼95 Å in DPC alone <em>versus</em> ∼142 Å in DMPC:DPC (1:2). SEC-MALLS shows that the molecular composition of the mTSPO belt is ∼98 molecules for DPC alone and ∼58 DMPC and ∼175 DPC for DMPC:DPC (1:2). Additionally, DMPC:DPC micelles stabilize mTSPO compared to DPC alone, where the protein has a greater propensity to aggregate. These structural changes are consistent with the increased affinity of mTSPO for the PK11195 ligand in presence of lipids (<em>K</em><sub>d</sub> ∼70 μM in DPC alone <em>versus</em> ∼0.91 μM in DMPC:DPC, 1:2), as measured by microscale thermophoresis (MST).</p><p>In conclusion, mixed lipid:surfactant micelles open new possibilities for the stabilization of membrane proteins and for their study in solution in a more biomimetic amphiphilic environment.</p></div>\",\"PeriodicalId\":251,\"journal\":{\"name\":\"Biochimie\",\"volume\":\"224 \",\"pages\":\"Pages 3-15\"},\"PeriodicalIF\":3.3000,\"publicationDate\":\"2024-04-23\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.sciencedirect.com/science/article/pii/S030090842400083X/pdfft?md5=9e427a6cb180bd19b969cc361f24fb0b&pid=1-s2.0-S030090842400083X-main.pdf\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Biochimie\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S030090842400083X\",\"RegionNum\":3,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"BIOCHEMISTRY & MOLECULAR BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biochimie","FirstCategoryId":"99","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S030090842400083X","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0

摘要

TSPO 是一种无处不在的跨膜蛋白,在神经成像中被用作药理学标记。哺乳动物 TSPO 的唯一已知原子结构来自与 PK11195 配体结合并处于 DPC 表面活性剂环境中的小鼠 TSPO(mTSPO)的溶液 NMR。我们测量了不同两亲环境对不含配体的 mTSPO 的影响,以研究其结构/功能并找到最佳溶解条件。通过用不同比例(0:1、1:2 和 2:1,w:w)的混合脂质:表面活性剂(DMPC:DPC)胶束取代纯化重组蛋白的 SDS 表面活性剂,mTSPO 的 α-螺旋含量和相互作用以及固有色氨酸(Trp)荧光逐渐增加:小角 X 射线散射(SAXS)显示,加入脂质后,mTSPO/带状复合物的扩展性更强:单独 DPC 中的 Dmax ∼95 Å,而 DMPC:DPC (1:2) 中的 Dmax ∼142 Å。SEC-MALLS 显示,单独使用 DPC 时,mTSPO 带的分子组成为 98 个分子,而使用 DMPC:DPC (1:2) 时,mTSPO 带的分子组成为 58 个 DMPC 和 175 个 DPC。此外,与单独使用 DPC 相比,DMPC:DPC 胶束能稳定 mTSPO,因为 DMPC:DPC 中的蛋白质更容易聚集。总之,脂质:表面活性剂混合胶束为稳定膜蛋白以及在更具生物仿真性的两亲环境中研究膜蛋白的溶液提供了新的可能性。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Enhanced structure/function of mTSPO translocator in lipid:surfactant mixed micelles

Enhanced structure/function of mTSPO translocator in lipid:surfactant mixed micelles

Enhanced structure/function of mTSPO translocator in lipid:surfactant mixed micelles

TSPO is a ubiquitous transmembrane protein used as a pharmacological marker in neuroimaging. The only known atomic structure of mammalian TSPOs comes from the solution NMR of mouse TSPO (mTSPO) bound to the PK11195 ligand and in a DPC surfactant environment. No structure is available in a biomimetic environment and without PK11195 which strongly stiffens the protein.

We measured the effect of different amphiphilic environments on ligand-free mTSPO to study its structure/function and find optimal solubilization conditions. By replacing the SDS surfactant, where the recombinant protein is purified, with mixed lipid:surfactant (DMPC:DPC) micelles at different ratios (0:1, 1:2, and 2:1, w:w), the α-helix content and interactions and the intrinsic tryptophan (Trp) fluorescence of mTSPO are gradually increased.

Small-angle X-ray scattering (SAXS) shows a more extended mTSPO/belt complex with the addition of lipids: Dmax ∼95 Å in DPC alone versus ∼142 Å in DMPC:DPC (1:2). SEC-MALLS shows that the molecular composition of the mTSPO belt is ∼98 molecules for DPC alone and ∼58 DMPC and ∼175 DPC for DMPC:DPC (1:2). Additionally, DMPC:DPC micelles stabilize mTSPO compared to DPC alone, where the protein has a greater propensity to aggregate. These structural changes are consistent with the increased affinity of mTSPO for the PK11195 ligand in presence of lipids (Kd ∼70 μM in DPC alone versus ∼0.91 μM in DMPC:DPC, 1:2), as measured by microscale thermophoresis (MST).

In conclusion, mixed lipid:surfactant micelles open new possibilities for the stabilization of membrane proteins and for their study in solution in a more biomimetic amphiphilic environment.

求助全文
通过发布文献求助,成功后即可免费获取论文全文。 去求助
来源期刊
Biochimie
Biochimie 生物-生化与分子生物学
CiteScore
7.20
自引率
2.60%
发文量
219
审稿时长
40 days
期刊介绍: Biochimie publishes original research articles, short communications, review articles, graphical reviews, mini-reviews, and hypotheses in the broad areas of biology, including biochemistry, enzymology, molecular and cell biology, metabolic regulation, genetics, immunology, microbiology, structural biology, genomics, proteomics, and molecular mechanisms of disease. Biochimie publishes exclusively in English. Articles are subject to peer review, and must satisfy the requirements of originality, high scientific integrity and general interest to a broad range of readers. Submissions that are judged to be of sound scientific and technical quality but do not fully satisfy the requirements for publication in Biochimie may benefit from a transfer service to a more suitable journal within the same subject area.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
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
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
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 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术官方微信