Joel Lapin, Emmanuel O. Awosanya, Richard J.A. Esteves, Alexander A. Nevzorov
{"title":"1H/13C/15N triple-resonance experiments for structure determinaton of membrane proteins by oriented-sample NMR","authors":"Joel Lapin, Emmanuel O. Awosanya, Richard J.A. Esteves, Alexander A. Nevzorov","doi":"10.1016/j.ssnmr.2020.101701","DOIUrl":null,"url":null,"abstract":"<div><p>The benefits of triple-resonance experiments for structure determination of macroscopically oriented membrane proteins by solid-state NMR are discussed. While double-resonance <sup>1</sup>H/<sup>15</sup><span>N experiments are effective for structure elucidation of alpha-helical domains, extension of the method of oriented samples to more complex topologies and assessing side-chain conformations necessitates further development of triple-resonance (</span><sup>1</sup>H/<sup>13</sup>C/<sup>15</sup>N) NMR pulse sequences. Incorporating additional spectroscopic dimensions involving <sup>13</sup>C spin-bearing nuclei, however, introduces essential complications arising from the wide frequency range of the <sup>1</sup>H-<sup>13</sup><span>C dipolar couplings and </span><sup>13</sup><span>C CSA (>20 kHz), and the presence of the </span><sup>13</sup>C-<sup>13</sup>C homonuclear dipole-dipole interactions. The recently reported ROULETTE–CAHA pulse sequence, in combination with the selective z-filtering, can be used to evolve the structurally informative <sup>1</sup>H-<sup>13</sup><span>C dipolar coupling arising from the aliphatic carbons while suppressing the signals from the carbonyl and methyl regions. Proton-mediated magnetization transfer under mismatched Hartman-Hahn conditions (MMHH) can be used to correlate </span><sup>13</sup>C and <sup>15</sup>N nuclei in such triple-resonance experiments for the subsequent <sup>15</sup><span>N detection. The recently developed pulse sequences are illustrated for n-acetyl Leucine<span> (NAL) single crystal and doubly labeled Pf1 coat protein reconstituted in magnetically aligned bicelles. An interesting observation is that in the case of </span></span><sup>15</sup>N-labeled NAL measured at <sup>13</sup>C natural abundance, the triple (<sup>1</sup>H/<sup>13</sup>C/<sup>15</sup>N) MMHH scheme predominantly gives rise to long-range intermolecular magnetization transfers from <sup>13</sup>C to <sup>15</sup>N spins; whereas direct Hartmann-Hahn <sup>13</sup>C/<sup>15</sup>N transfer is entirely intramolecular. The presented developments advance NMR of oriented samples for structure determination of membrane proteins and liquid crystals.</p></div>","PeriodicalId":21937,"journal":{"name":"Solid state nuclear magnetic resonance","volume":null,"pages":null},"PeriodicalIF":1.8000,"publicationDate":"2021-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1016/j.ssnmr.2020.101701","citationCount":"1","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Solid state nuclear magnetic resonance","FirstCategoryId":"92","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0926204020300631","RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"CHEMISTRY, PHYSICAL","Score":null,"Total":0}
引用次数: 1
Abstract
The benefits of triple-resonance experiments for structure determination of macroscopically oriented membrane proteins by solid-state NMR are discussed. While double-resonance 1H/15N experiments are effective for structure elucidation of alpha-helical domains, extension of the method of oriented samples to more complex topologies and assessing side-chain conformations necessitates further development of triple-resonance (1H/13C/15N) NMR pulse sequences. Incorporating additional spectroscopic dimensions involving 13C spin-bearing nuclei, however, introduces essential complications arising from the wide frequency range of the 1H-13C dipolar couplings and 13C CSA (>20 kHz), and the presence of the 13C-13C homonuclear dipole-dipole interactions. The recently reported ROULETTE–CAHA pulse sequence, in combination with the selective z-filtering, can be used to evolve the structurally informative 1H-13C dipolar coupling arising from the aliphatic carbons while suppressing the signals from the carbonyl and methyl regions. Proton-mediated magnetization transfer under mismatched Hartman-Hahn conditions (MMHH) can be used to correlate 13C and 15N nuclei in such triple-resonance experiments for the subsequent 15N detection. The recently developed pulse sequences are illustrated for n-acetyl Leucine (NAL) single crystal and doubly labeled Pf1 coat protein reconstituted in magnetically aligned bicelles. An interesting observation is that in the case of 15N-labeled NAL measured at 13C natural abundance, the triple (1H/13C/15N) MMHH scheme predominantly gives rise to long-range intermolecular magnetization transfers from 13C to 15N spins; whereas direct Hartmann-Hahn 13C/15N transfer is entirely intramolecular. The presented developments advance NMR of oriented samples for structure determination of membrane proteins and liquid crystals.
期刊介绍:
The journal Solid State Nuclear Magnetic Resonance publishes original manuscripts of high scientific quality dealing with all experimental and theoretical aspects of solid state NMR. This includes advances in instrumentation, development of new experimental techniques and methodology, new theoretical insights, new data processing and simulation methods, and original applications of established or novel methods to scientific problems.