Sensitivity Enhancement and Contrasting Information Provided by Free Radicals in Oriented-Sample NMR of Bicelle-Reconstituted Membrane Proteins
 

Sensitivity Enhancement and Contrasting Information Provided by Free Radicals in Oriented-Sample NMR of Bicelle-Reconstituted Membrane Proteins

Publication date: Available online 28 November 2013
Source:Journal of Magnetic Resonance</br>
Author(s): Deanna M. Tesch , Alexander A. Nevzorov</br>
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 the their effect on the longitudinal relaxation times (T 1Z) was investigated. The dramatically shortened T 1Z’s allowed for the signal gain per unit time to be used for either: i) up to a three-fold 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 “optimal” 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.
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