Characterization of Membrane Protein‐Lipid Interactions in Unfolded OmpX with Enhanced Time Resolution by Hyperpolarized NMR

Abstract

Proton nuclear spins of dodecyl phosphocholine molecules below the critical micelle concentration are hyperpolarized by using dissolution dynamic nuclear polarization (D-DNP). NMR signal enhancements of 1210 � 400 and 1610 � 550 are obtained at 9.4 T, for choline methyls in the head group of the lipid and for the tail-end methyl group, respectively. This polarization is transferred to the unfolded protein through the nuclear Overhauser effect, after dilution to a final denaturant concentration of 0.8 M urea. As a result, the amide and aromatic side-chain signals of the protein are increased up to sixfold. Selective inversion pulses applied either on the head-group or tail-group of the lipid are used to identify the source of the transferred polarization. The normalized cross-relaxation rates of σ N,tail = À 1.8 � 0.1 s À 1 M À 1 and σ N,head = À 0.5 � 0.3 s À 1 M À 1 are obtained, showing a larger polarization transfer from the tail groups. These cross-relaxation rates are determined at a low urea concentration, which constitutes refolding conditions for the protein. The sensitivity enhancement by D-DNP permits to access these conditions with a measurement time on the order of seconds, and may further open the possibility to investigate structural changes in membrane proteins during folding.

Selective Isotope Labeling and LC-Photo-CIDNP Enable NMR Spectroscopy at Low-Nanomolar Concentration

“…The well-known dissolution DNP (dDNP) technology enabled the detection of 20 μM protein samples . This technology has also been used in the context of protein–ligand interactions. ,− In addition, Kouřil et al recently showed that 32% 13 C polarization was rendered possible by bullet DNP, an interesting variation of dDNP employing small sample-dilution factors during the dissolution process. In both the above studies, considerably higher sample concentrations were required than in the present work.…”

Challenges and Advances in the Application of Dynamic Nuclear Polarization to Liquid-State NMR Spectroscopy

“…An advantage of DNP NMR is that, by using site-directed spin labeling, hyperpolarization can be achieved in a site-specific manner, allowing highly localized probing of protein dynamics and secondary structure. Hilty et al recently used the method of dissolution-DNP (d-DNP) to hyperpolarize lipids (dodecyl phosphocholine) and monitored lipid interactions with the membrane protein OmpX . By exploiting the facile exchange of amide protons, hyperpolarized water has also been used to obtain high-resolution 2D NMR spectra of disordered proteins .…”

“…Hilty et al recently used the method of dissolution-DNP (d-DNP) to hyperpolarize lipids (dodecyl phosphocholine) and monitored lipid interactions with the membrane protein OmpX. 47 By exploiting the facile exchange of amide protons, hyperpolarized water has also been used to obtain high-resolution 2D NMR spectra of disordered proteins. 48 Hyperpolarized ligands have been used to selectively enhance signals of ligand-binding sites 49,50 or to transfer polarization to other nearby ligands 51 on the protein.…”