期刊
JOURNAL OF BIOMOLECULAR NMR
卷 73, 期 10-11, 页码 617-624出版社
SPRINGER
DOI: 10.1007/s10858-019-00273-1
关键词
Oriented-sample solid-state NMR; Paramagnetic relaxation enhancement; Heat compensation; SAMPI4; Membrane proteins; Sarcolipin; Bicelles
资金
- NHLBI NIH HHS [R01 HL144130] Funding Source: Medline
- NIGMS NIH HHS [R01 GM064742] Funding Source: Medline
- NIH HHS [GM 64742, HL 144130] Funding Source: Medline
Oriented sample solid-state NMR (OS-ssNMR) spectroscopy is a powerful technique to determine the topology of membrane proteins in oriented lipid bilayers. Separated local field (SLF) experiments are central to this technique as they provide first-order orientational restraints, i.e., dipolar couplings and anisotropic chemical shifts. Despite the use of low-E (or E-free) probes, the heat generated during the execution of 2D and 3D SLF pulse sequences causes sizeable line-shape distortions. Here, we propose a new heat-compensated SE-SAMPI4 (hcSE-SAMPI4) pulse sequence that holds the temperature constant for the duration of the experiment. This modification of the SE-SAMPI4 results in sharper and more intense resonances without line-shape distortions. The spectral improvements are even more apparent when paramagnetic relaxation agents are used to speed up data collection. We tested the hcSE-SAMPI4 pulse sequence on a single-span membrane protein, sarcolipin (SLN), reconstituted in magnetically aligned lipid bicelles. In addition to eliminating peak distortions, the hcSE-SAMPI4 experiment increased the average signal-to-noise ratio by 20% with respect to the original SE-SAMPI4.
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