{"title":"调整固态 DNP 方法以研究 α-synuclein LLPS。","authors":"Ribal Jabbour , Sreejith Raran-Kurussi , Vipin Agarwal , Asif Equbal","doi":"10.1016/j.bpc.2024.107303","DOIUrl":null,"url":null,"abstract":"<div><p>Dynamic Nuclear Polarization (DNP) is a technique that leverages the quantum sensing capability of electron spins to enhance the sensitivity of nuclear magnetic resonance (NMR) signals, especially for insensitive samples. Glassing agents play a crucial role in the DNP process by facilitating the transfer of polarization from the unpaired electron spins to the nuclear spins along with cryoprotection of biomolecules. <span><math><mi>DNP</mi><mspace></mspace><mi>juice</mi></math></span> comprising of glycerol-<span><math><msub><mi>d</mi><mn>8</mn></msub></math></span>/<span><math><msub><mi>D</mi><mn>2</mn></msub><mi>O</mi></math></span>/<span><math><msub><mi>H</mi><mn>2</mn></msub><mi>O</mi></math></span> has been extensively used for this purpose over the past two decades. Polyethylene glycol (PEG), also used as a cryoprotectant, is often used as a crowding agent in experimental setups to mimic cellular conditions, particularly the <span><math><mi>in</mi><mspace></mspace><mi>vitro</mi></math></span> preparation of liquid-liquid phase separated (LLPS) condensates. In this study, we investigate the efficacy of PEG as an alternative to glycerol in the <em>DNP juice</em>, critical for signal enhancement. The modified DNP matrix leads to high DNP enhancement which enables direct study of LLPS condensates by solid-state DNP methods without adding any external constituents. An indirect advantage of employing PEG is that the PEG signals appear at <span><math><mo>∼</mo></math></span>72.5 ppm and are relatively well-separated from the aliphatic region of the protein spectra. Large cross-effect DNP enhancement is attained for <span><math><msup><mrow></mrow><mn>13</mn></msup><mi>C</mi></math></span>-glycine by employing the PEG-water mixture as a glassing agent and ASYMPOL-POK as the state-of-art polarizing agent, without any deuteration. The DNP enhancement and the buildup rates are similar to results obtained with <em>DNP juice</em>, conforming to that PEG serves as a good candidate for both inducing crowding and glassing agent in the study of LLPS.</p></div>","PeriodicalId":8979,"journal":{"name":"Biophysical chemistry","volume":"313 ","pages":"Article 107303"},"PeriodicalIF":3.3000,"publicationDate":"2024-07-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Tailoring solid-state DNP methods to the study of α-synuclein LLPS\",\"authors\":\"Ribal Jabbour , Sreejith Raran-Kurussi , Vipin Agarwal , Asif Equbal\",\"doi\":\"10.1016/j.bpc.2024.107303\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>Dynamic Nuclear Polarization (DNP) is a technique that leverages the quantum sensing capability of electron spins to enhance the sensitivity of nuclear magnetic resonance (NMR) signals, especially for insensitive samples. Glassing agents play a crucial role in the DNP process by facilitating the transfer of polarization from the unpaired electron spins to the nuclear spins along with cryoprotection of biomolecules. <span><math><mi>DNP</mi><mspace></mspace><mi>juice</mi></math></span> comprising of glycerol-<span><math><msub><mi>d</mi><mn>8</mn></msub></math></span>/<span><math><msub><mi>D</mi><mn>2</mn></msub><mi>O</mi></math></span>/<span><math><msub><mi>H</mi><mn>2</mn></msub><mi>O</mi></math></span> has been extensively used for this purpose over the past two decades. Polyethylene glycol (PEG), also used as a cryoprotectant, is often used as a crowding agent in experimental setups to mimic cellular conditions, particularly the <span><math><mi>in</mi><mspace></mspace><mi>vitro</mi></math></span> preparation of liquid-liquid phase separated (LLPS) condensates. In this study, we investigate the efficacy of PEG as an alternative to glycerol in the <em>DNP juice</em>, critical for signal enhancement. The modified DNP matrix leads to high DNP enhancement which enables direct study of LLPS condensates by solid-state DNP methods without adding any external constituents. An indirect advantage of employing PEG is that the PEG signals appear at <span><math><mo>∼</mo></math></span>72.5 ppm and are relatively well-separated from the aliphatic region of the protein spectra. Large cross-effect DNP enhancement is attained for <span><math><msup><mrow></mrow><mn>13</mn></msup><mi>C</mi></math></span>-glycine by employing the PEG-water mixture as a glassing agent and ASYMPOL-POK as the state-of-art polarizing agent, without any deuteration. The DNP enhancement and the buildup rates are similar to results obtained with <em>DNP juice</em>, conforming to that PEG serves as a good candidate for both inducing crowding and glassing agent in the study of LLPS.</p></div>\",\"PeriodicalId\":8979,\"journal\":{\"name\":\"Biophysical chemistry\",\"volume\":\"313 \",\"pages\":\"Article 107303\"},\"PeriodicalIF\":3.3000,\"publicationDate\":\"2024-07-30\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Biophysical chemistry\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S0301462224001327\",\"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":"Biophysical chemistry","FirstCategoryId":"99","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0301462224001327","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}
Tailoring solid-state DNP methods to the study of α-synuclein LLPS
Dynamic Nuclear Polarization (DNP) is a technique that leverages the quantum sensing capability of electron spins to enhance the sensitivity of nuclear magnetic resonance (NMR) signals, especially for insensitive samples. Glassing agents play a crucial role in the DNP process by facilitating the transfer of polarization from the unpaired electron spins to the nuclear spins along with cryoprotection of biomolecules. comprising of glycerol-// has been extensively used for this purpose over the past two decades. Polyethylene glycol (PEG), also used as a cryoprotectant, is often used as a crowding agent in experimental setups to mimic cellular conditions, particularly the preparation of liquid-liquid phase separated (LLPS) condensates. In this study, we investigate the efficacy of PEG as an alternative to glycerol in the DNP juice, critical for signal enhancement. The modified DNP matrix leads to high DNP enhancement which enables direct study of LLPS condensates by solid-state DNP methods without adding any external constituents. An indirect advantage of employing PEG is that the PEG signals appear at 72.5 ppm and are relatively well-separated from the aliphatic region of the protein spectra. Large cross-effect DNP enhancement is attained for -glycine by employing the PEG-water mixture as a glassing agent and ASYMPOL-POK as the state-of-art polarizing agent, without any deuteration. The DNP enhancement and the buildup rates are similar to results obtained with DNP juice, conforming to that PEG serves as a good candidate for both inducing crowding and glassing agent in the study of LLPS.
期刊介绍:
Biophysical Chemistry publishes original work and reviews in the areas of chemistry and physics directly impacting biological phenomena. Quantitative analysis of the properties of biological macromolecules, biologically active molecules, macromolecular assemblies and cell components in terms of kinetics, thermodynamics, spatio-temporal organization, NMR and X-ray structural biology, as well as single-molecule detection represent a major focus of the journal. Theoretical and computational treatments of biomacromolecular systems, macromolecular interactions, regulatory control and systems biology are also of interest to the journal.