Lipid-protein correlations in nanoscale phospholipid bilayers determined by solid-state nuclear magnetic resonance

Abstract

Nanodiscs are examples of discoidal nanoscale lipid-protein particles that have been extremely useful for the biochemical and biophysical characterization of membrane proteins. They are discoidal lipid bilayer fragments encircled and stabilized by two amphipathic helical proteins named membrane scaffolding protein (MSP), ~10 nm in size. Nanodiscs are homogeneous, easily prepared with reproducible success, amenable to preparations with a variety of lipids, and stable over a range of temperatures. Here we present solid-state nuclear magnetic resonance (SSNMR) studies on lyophilized, rehydrated POPC Nanodiscs prepared with uniformly (13)C-, (15)N-labeled MSP1D1 (Δ1-11 truncated MSP). Under these conditions, by SSNMR we directly determine the gel-to-liquid crystal lipid phase transition to be at 3 ± 2 °C. Above this phase transition, the lipid (1)H signals have slow transverse relaxation, enabling filtering experiments as previously demonstrated for lipid vesicles. We incorporate this approach into two- and three-dimensional heteronuclear SSNMR experiments to examine the MSP1D1 residues interfacing with the lipid bilayer. These (1)H-(13)C and (1)H-(13)C-(13)C correlation spectra are used to identify and quantify the number of lipid-correlated and solvent-exposed residues by amino acid type, which furthermore is compared with molecular dynamics studies of MSP1D1 in Nanodiscs. This study demonstrates the utility of SSNMR experiments with Nanodiscs for examining lipid-protein interfaces and has important applications for future structural studies of membrane proteins in physiologically relevant formulations.

Figure 1. Gel to liquid crystalline transition of lipids in Nanodiscs

(a) A cartoon representation of a Nanodisc with MSP1D1 in red and POPC headgroups in green and tails in white. ¹H 1D spectra of POPC Nanodiscs at (b) 12 °C and (c) -20 °C. Spectra were acquired on a 600 MHz (¹H frequency) spectrometer. The acyl ¹H linewidth and peak height indicates the phase transition. (d) Graph of T₂ values as a function of temperature for the acyl methylene ¹H signals (1.3 ppm). The lipid phase transition in POPC Nanodiscs is determined to be at +3 °C.

Figure 2. ¹H-¹³C-¹³C 3D pulse sequence used to obtain lipid to protein correlations

Following presaturation of ¹³C Boltzmann polarization, mobile ¹H signals are selected by a T₂ filter. ¹H polarization is then transferred by spin diffusion to immobile protons, and to the attached ¹³C by cross polarization. Following evolution of the ¹³C chemical shifts, ¹³C-¹³C mixing is achieved by SPC-5 (84), and signals detected under TPPM decoupling (72).

Figure 3. ¹³C 1D and ¹H-¹³C 2D spectra of POPC bilayer in a Nanodisc

(a) 1D ¹³C spectrum of Nanodiscs with uniformly-¹³C,¹⁵N-labeled MSP1D1 and natural abundance POPC lipids at 8 °C on a 600 MHz (¹H frequency) spectrometer. The lipid spectrum was acquired with a 1 ms T₂ filter, no ¹H-¹H mixing and 0.4 ms ¹H-¹³C contact time. The data was processed with 25 Hz Lorentzian-to-Gaussian line-broadening. (b) ¹H-¹³C 2D spectrum of the same sample acquired under the same conditions for 6.5 hrs, with a schematic of a POPC lipid molecule. The spectrum was processed with 100 Hz of Lorentzian-to-Gaussian line broadening in each dimension.

Figure 4. ¹³C 1D spectra of MSP1D1 in POPC Nanodiscs

Spectra were acquired on U-¹³C,¹⁵N-labeled MSP1D1 at 8 °C. ¹³C 1D spectra were acquired with a 1 ms T₂ filer, a 0.4 ms HC contact time, where ¹H-¹H mixing time was incremented and sequentially set to 3 μs, 1 ms, 3 ms, 10 ms and 30 ms. The data were processed with 30 Hz Lorentzian-to-Gaussian line broadening.

Figure 5. ¹H-¹³C 2D spectra of MSP1D1 in POPC Nanodiscs

Experimental spectrum acquired on U-¹³C,¹⁵N-labeled MSP1D1 at 15 °C. Spectra were acquired with a 1 ms T₂ filer, a 0.4 ms ¹H-¹³C contact time, and a 30 ms ¹H-¹H mixing time. The spectrum was processed with back linear prediction, 100 Hz net line broadening in the direct, and 80 Hz in the indirect dimension (Lorentzian-to-Gaussian apodization), and zero filled to 4096 (ω₂) × 16 384 (ω₁) complex points before Fourier transformation. Data were acquired in ~7 hrs.

Figure 6. ¹H-¹³C-¹³C 3D spectrum of MSP1D1 in Nanodiscs

¹H-¹³C-¹³C spectrum acquired at 600 MHz (¹H frequency), 8 °C sample temp in ~115 hours. The T₂ filter time was set to 1 ms to eliminate protein ¹H polarization. A ¹H-¹H mixing time of 30 ms was used followed by ¹H-¹³C cross polariation. ¹³C-¹³C mixing with 12 ms of SPC-5 was used without the double quantum filiter. Water (a) and lipid (b) ¹³C-¹³C planes are show with peaks labeled by amino acid type (88). Positive contours shown in red, negative in blue. The seeming artifact in the lipid plane is due to a strong signal originating from lipids correlated to themselves.

Figure 7. Plot of Water to Lipid ratios determined by NMR and Molecular Dynamics

Ratio of CB carbons in MSP within 4.0 Å of a water molecule to the ratio of CB carbons within 4.0 Å of lipid plotted versus ratio of cross peak intensities for the same residues at the water and lipid ¹H frequencies in the ¹H-¹³C-¹³C 3D spectrum. MD ratios are the average number of CB atoms for a given residue within 4.0 Å of lipid or water averaged over two MSP molecules for the last 0.75 ns of a 4.5 ns molecular dynamics simulation. NMR ratios were calculated using integrated peak intensities for ¹³C-¹³C cross peaks corresponding to the same residues appearing at both the lipid and water ¹H frequency in the ¹H-¹³C-¹³C 3D spectrum. Error bars for the NMR ratio were calculated using the signal to noise of the peaks in the 3D using the method of Mani et al. (64). The linear best fit line is plotted. The NMR ratio is scaled due to different ¹H T₂ as well as differences in total initial polarization.