Sensitivity enhancement and contrasting information provided by free radicals in oriented-sample NMR of bicelle-reconstituted membrane proteins
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- 作者：Deanna M. Tesch ; Alex ; er A. Nevzorov
- 关键词：Membrane proteins ; Oriented samples ; OS-NMR ; Longitudinal relaxation time ; Paramagnetic relaxation enhancement ; PRE ; Free radicals ; Magnetically aligned bicelles
- 刊名：Journal of Magnetic Resonance
- 出版年：February, 2014
- 年：2014
- 卷：239
- 期：Complete
- 页码：9-15
- 全文大小：1743 K
文摘
Elucidating structure and topology of membrane proteins (MPs) is essential for unveiling functionality of these important biological constituents. Oriented-sample solid-state NMR (OS-NMR) is capable of providing such information on MPs under nearly physiological conditions. However, two dimensional OS-NMR experiments can take several days to complete due to long longitudinal relaxation times combined with the large number of scans to achieve sufficient signal sensitivity in biological samples. Here, free radicals 5-DOXYL stearic acid, TEMPOL, and CAT-1 were added to uniformly 15N-labeled Pf1 coat protein reconstituted in DMPC/DHPC bicelles, and their effect on the longitudinal relaxation times (T1Z) was investigated. The dramatically shortened T1Z鈥檚 allowed for the signal gain per unit time to be used for either: (i) up to a threefold reduction of the total experimental time at 99% magnetization recovery or (ii) obtaining up to 74% signal enhancement between the control and radical samples during constant experimental time at 鈥渙ptimal鈥?relaxation delays. In addition, through OS-NMR and high-field EPR studies, free radicals were able to provide positional constraints in the bicelle system, which provide a description of the location of each residue in Pf1 coat protein within the bicellar membranes. This information can be useful in the determination of oligomerization states and immersion depths of larger membrane proteins.