Solid-state NMR spectroscopy of 18.5 kDa myelin basic protein reconstituted with lipid vesicles: spectroscopic characterisation and spectral assignments of solvent-exposed protein fragments

Abstract

Myelin basic protein (MBP, 18.5 kDa isoform) is a peripheral membrane protein that is essential for maintaining the structural integrity of the multilamellar myelin sheath of the central nervous system. Reconstitution of the most abundant 18.5 kDa MBP isoform with lipid vesicles yields an aggregated assembly mimicking the protein's natural environment, but which is not amenable to standard solution NMR spectroscopy. On the other hand, the mobility of MBP in such a system is variable, depends on the local strength of the protein-lipid interaction, and in general is of such a time scale that the dipolar interactions are averaged out. Here, we used a combination of solution and solid-state NMR (ssNMR) approaches: J-coupling-driven polarization transfers were combined with magic angle spinning and high-power decoupling to yield high-resolution spectra of the mobile fragments of 18.5 kDa murine MBP in membrane-associated form. To partially circumvent the problem of short transverse relaxation, we implemented three-dimensional constant-time correlation experiments (NCOCX, NCACX, CONCACX, and CAN(CO)CX) that were able to provide interresidue and intraresidue backbone correlations. These experiments resulted in partial spectral assignments for mobile fragments of the protein. Additional nuclear Overhauser effect spectroscopy (NOESY)-based experiments revealed that the mobile fragments were exposed to solvent and were likely located outside the lipid bilayer, or in its hydrophilic portion. Chemical shift index analysis showed that the fragments were largely disordered under these conditions. These combined approaches are applicable to ssNMR investigations of other peripheral membrane proteins reconstituted with lipids.

Fig. 1

Experimental pulse sequences. In all pulse sequences, narrow rectangles represent 90° pulses, while wide rectangles are 180° pulses. (A) The HCC three-dimensional experiment. Phase cycling in the experiment was as follows: ϕ₁ = ϕ₂ = ϕ₄ = x, ϕ₃ =(y, −y), ϕ₅ = ϕ₆ = y, ϕ₇ =(8(x), 8(−x)), ϕ₈ =(2(x), 2(y), 2(−x), 2(−y)), ϕ_rec =(x, −x, y, −y, −x, x, −y, y, −x, x, −y, y, x, −x, y, −y). The INEPT periods were as follows: τ₁ =1.4 ms, τ₂ =0.9 ms. The ¹³C carrier frequency was placed at 45 ppm for mixing between aliphatic carbons, and at 75 ppm for mixing between aliphatic and aromatic carbons. (B) The HHCC NOE experiment used to probe through-space ¹H–¹H interactions. Phase cycling was the same as in panel A, with two additional pulses phase-cycled to eliminate T₁ relaxation effects during NOE mixing: ϕ₁₀ =(−x, x), ϕ₁₁ =(x, −x). (C) Pulse sequence for the three-dimensional constant-time NCACX/NCOCX experiments. The INEPT periods τ₃ and τ₄ were 1.7 ms. Selective pulses were implemented as a Gaussian cascade [69] of 200 μs for C^α, and as a single Gaussian pulse of 300 μs for carbonyl atoms. The value of ε₁ was equal to the length of the selective pulse ϕ₇ = ϕ₁₇. Time constants T₁ and T₂ were set to 21 ms and 25 ms, respectively, in the NCACX experiment, and 24 ms (both) in the NCOCX experiment. Phase cycling in this experiment was as follows: ϕ₁ = ϕ₂ = ϕ₄ = ϕ₅ = ϕ₆ = ϕ₇ = ϕ₈ = ϕ₉ = ϕ₁₀ = ϕ₁₁ = ϕ₁₂ = ϕ₁₇ = x, ϕ₃ = (y, −y), ϕ₁₃ = ϕ₁₈ = y, ϕ₁₉ = (2(y), 2(−x), 2(−y), 2(x)), ϕ_rec =(x, −x, y, −y, −x, x, −y, y). The pulse sequence and the basic principles in the NCACX experiment were very similar to those of the NCOCX experiment, except that the selective pulses applied to the ¹³C′ and ¹³C^α spins were switched. In addition, the ¹³C^α chemical shift evolution dimension was kept less than 10 ms ( $<1/(2{\text{J}}_{\text{CC}}^{\alpha \beta })$), to minimise the effect of J-coupling between ¹³C^α spins and other aliphatic spins. The selective pulses used on ¹³C^α and ¹³C′ were implemented as Gaussian cascades [69] and Gaussian pulses, respectively. (D) Pulse sequence for the three-dimensional constant-time CONCACX/CAN(CO)CX experiments. Time constants in CONCACX were T₁ =24 ms, T₂ =26 ms, T₃ =30 ms, τ₃ =10 ms, and in CAN(CO)CX they were T₁ =20 ms, T₂ =24 ms, T₃ =26 ms, τ₃ =15 ms. Here, ε₁ is the length of the selective pulse with phase ϕ₂₉, while ε₂ is the length of selective pulse with phase ϕ₇. The phase cycling in the pulse sequence was as follows: ϕ₁ = ϕ₂ = ϕ₄ = ϕ₆ = ϕ₇ = ϕ₈ = ϕ₉ = ϕ₁₀ = ϕ₁₂ = ϕ₁₇ = ϕ₂₃ = ϕ₂₈ = ϕ₂₉ = x, ϕ₃ = ϕ₁₈ = y, ϕ₅ =(x, −x), ϕ₁₁ =(2(x), 2(y), 2(−x), 2(−y)), ϕ₁₃ =(2(y), 2(−x), 2(−y), 2(x)), ϕ₂₅ = (−x), ϕ_rec =(x, −x, y, −y, −x, x, −y, y). GARP (globally optimised alternating phase rectangular pulses) [70] decoupling was used to remove NC J-couplings.

Fig. 2

Schematic representation of the four triple-resonance experiments used to obtain sequential assignments. Correlated residues are indicated by light gray boxes. Solid arrows show dominating one-bond polarization transfers, while dashed arrows show two-bond N[i]–C^α[i−1] transfers, which could not be observed in our spectra.

Fig. 3

The CPMAS carbon spectrum (A) and INEPT carbon spectrum (B) of fully ¹³C,¹⁵N-labelled 18.5 kDa rmMBP samples reconstituted with lipids. TPPM decoupling of 71.4 kHz was applied during acquisition. Both spectra were taken at 600 MHz, and at a temperature of 32 °C. The CPMAS spectrum shown in the image was collected with a contact time of 3 ms, and with 4180 scans, and at a spinning frequency of 20 kHz. Experiments with shorter cross-polarization mixing times and different cross-polarization power levels resulted in spectra of similar intensities. The INEPT spectrum was collected at 32 °C with 32 scans, at a spinning frequency of 10 kHz. The acquisition lengths were 21 ms and 40 ms in the CPMAS and INEPT experiments, respectively. All spectra were processed with exponential function apodisation of 10 Hz.

Fig. 4

A comparison of the effect of the decoupling on the resolution of ¹³C INEPT spectra. High resolution is seen even at low-power WALTZ-16 [53,54] decoupling, indicating the mobile nature of the residues contributing to the INEPT spectra. Even higher resolution is observed at moderate- and high-power TPPM decoupling. All experiments were obtained on a 600-MHz Bruker spectrometer with 10 kHz MAS. The acquisition and spectral processing parameters are given in the caption to Fig. 1.

Fig. 5

(A) The two-dimensional ¹H–¹³C HSQC ¹³C-detected spectrum, obtained using the pulse sequence shown in Fig. 1A, with t₂ evolution and TOBSY mixing set to zero. The total ¹H evolution time was 16 ms with 200 points. The spectrum was collected with 16 scans per point and with the recycling delay of 2 s. The spectrum was processed in NMRPipe [44], employing exponential function apodisation of 20 Hz in the direct dimension and cosine square apodisation in the indirect dimension. Data were zero filled up to 4096×2048 points in the direct and indirect dimensions, respectively. (B, C) Representative two-dimensional planes of the three-dimensional HCC chemical shift correlation experiment. The total ¹H evolution time was 8.2 ms with 88 points, while the total indirect carbon evolution time was 10 ms with 240 points. The spectrum was collected with 8 scans per point. The recycling delay was set to 1.7 s. The carrier frequency was set to 45 ppm. Both spectra were processed in NMRPipe [44], employing exponential function apodisation of 10 Hz in the direct dimension, and cosine square apodisation in the two indirect dimensions. Data were zero filled up to 4096×2048×2048 points in the direct and two indirect dimensions, respectively.

Fig. 6

The two-dimensional ¹³C–¹³C planes of (A) the three-dimensional NCOCX, and (B) NCACX experiments. In the NCOCX experiment, a total of 120 t₁ points were collected, with a maximum evolution time of 12.3 ms. A total of 100 points were taken in t₂, resulting in the total evolution time of 11 ms. In the NCACX experiment, the t₁ evolution time was 9.6 ms with 96 points, and the t₂ evolution time was 8.3 ms with 100 points. TOBSY mixing times of 9.9 ms and 7.5 ms were used in the NCOCX and NCACX experiments, respectively. The number of scans per free induction decay (FID) was 16 in the NCOCX and 24 in the NCACX experiments. The carrier frequency was set at 175 ppm for carbon in NCOCX, at 50 ppm for carbon in NCACX, and at 116 ppm for nitrogen. The recycling delay was set to 2 s. A 50 kHz TPPM decoupling was applied on the ¹H channel during ¹³C′–¹⁵N/¹³C^α–¹⁵N polarization transfers, and during direct and indirect detections.

Fig. 7

Strip plots from the three-dimensional NCOCX, NCACX, and CONCACX experiments, showing the D143-T147 amino acid stretch. The NCOCX experiment establishes N[i+1]–C′ [i]–C^α[i]/C^β[i] correlations shown in green, whereas the CONCACX experiment gives correlations between C′ [i], N[i+1], and C^α[i+1] spins, indicated in red. Shared C′[i] and N[i+1] shifts allow one to establish interresidue C^α[i]–C^α[i+1] correlations, and “walk” sequentially along the backbone, as shown by the horizontal and vertical dashed lines. Carbonyl shifts for each strip are shown on the top. The NCACX slice for T147 is shown in blue. The NCOCX and NCACX spectra were acquired as described in the caption of Fig. 6. The CONCACX spectrum was acquired with total t₁ and t₂ acquisition lengths of 11.4 ms and 10 ms, respectively, with 92 and 96 points in the carbonyl and nitrogen indirect dimensions, respectively. TPPM decoupling of 50 kHz was applied during NC INEPT transfers and direct acquisition. The number of scans per FID was 16, with a recycling delay of 2 s. The CAN(CO)CX experiment was collected with similar parameters, except for TOBSY mixing, which was 6 ms.

Fig. 8

A comparison of the two-dimensional (ω₁-¹H, ω₃-¹³C) projections of the (A) HCC, and (B) HHCC experiments. A series of extra peaks appearing at 4.6 ppm (the ¹H frequency of H₂O) is indicated by the arrows in panel B. The NOE mixing time was 50 ms.

Fig. 9

(A) Chemical shift index analysis of C^α of the backbone assignments. All of the random coil chemical shifts are sequence-corrected based on [67]. Secondary chemical shifts for tentatively assigned residues are shown in gray. (B) The ¹⁵N linewidths for assigned residues. Linewidths for tentatively assigned residues are shown in gray.