1. IntroductionMembrane proteins make up approximately one-third of the total number of known proteins [27]. They play several significant roles in biological systems that includes transporting ions, acting as receptors, participating in membrane fusion and destabilization, and many others. Despite the abundance and clear importance of membrane-associated proteins, limited information about these systems exists. Structural studies of these membrane proteins are key to understand their biological functions. X-ray crystallography has been used to elucidate structural information of biologically significant protein systems [28-35]. However, the hydrophobic surfaces associated with membrane-bound protein systems make the crystallization process very challenging. Although investigators are making progress with X-ray techniques, still only a limited list of membrane protein structures have been obtain via X-ray crystallography [36]. Alternatively, solid-state NMR spectroscopy is a powerful technique that can be used to provide structural, orientational, and dynamic information about membrane protein systems in model membrane systems [2,37,38].
2. Solid-State NMR Approaches2.1. <sup>2</sup>H Solid-State NMR Spectroscopy of Lipids with<sup> 2</sup>H-Lableled Acyl ChainsMultilamellar vesicles (MLVs) can be prepared from phospholipids such as 1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine (POPC) and the details of sample preparation and type of lipid was reported previously [3-7]. Approximately 10% of deuterated POPC (sn-1 chain, POPC-d31, see Figure 1) or any other deuterated lipid of interest can be added to each sample. Other lipids can be used such as the negatively charged lipids such as 1-palmitoyl(d31)-2-oleoyl-sn-glycero-3-[phospho-L-serine] phospholipids (POPS-d31) used in Sap C studies (see Figure 2" target="_self"> Figure 2) and Distearoyl-d70-phosphatidylglycerol (DSPGd70) (see Figure 3).
The effect of embedded proteins on the order and dynamics of the acyl chains of POPC-d31 bilayers can be investigated utilizing 2H solid-state NMR spectroscopy in the absence as well as in the presence of (X) mol% of the protein with respect to the lipids at different temperatures [40]. For example, in Figure 4(a), the central resonance doublet corresponds to the terminal CD3 groups (Carbon # 1 in Figure 1) and the remaining over-lapped doublets result from the different CD2 segments of the acyl chain of POPC-d31. The addition of (X) mol% of specific protein with respect to the lipids to POPC MLVs could alter (depending on the protein of interest) the lineshape and the spectral resolution of the 2H NMR spectra (Figure 4(b)) when compared to the control (Figure 4(a)) sample.
In Figure 4(b), the loss in spectral resolution is manifested by the disappearance of sharp edges of the 2H NMR peaks. The changes in spectral resolution of the 2H NMR spectra indicate that the protein interacts with the POPC-d31-containing MLVs when compared to the control. If the spectral resolution and the line shapes did not change when compared to the control, this indicates that the protein does not interact significantly with the POPC MLVs as indicated in a previous study from the Lorigan Lab using the transmembrane domain segment of WTPLB [12].
All the powder-type 2H NMR spectra of multilamellar dispersions of POPC-d31 can be numerically deconvoluted (dePaked) using the algorithm of McCabe and Wassal [41,42]. These spectra can be dePaked such that the bilayer normal was perpendicular with respect to the direction of the static magnetic field. Then, the quadrupolar splittings can be directly measured from the dePaked spectra and converted into order parameters as described before [11,12]. The quadrupolar splittings of the CD3 methyl groups (see Figure 1) at the end of the acyl chains are the smallest and close to 0 kHz because
they rotate at the fastest frequency. The second smallest splitting was assigned to the 2H attached to C-14 and so forth along the acyl chain. The quadrupolar splittings for the deuterons in the plateau region were estimated by integration of the last broad peak.
The quadrupolar splittings can be directly measured from the dePaked spectra and converted into the SCD order parameter using the following expression; 
= 3/4(e2qQ/h)
[43-45]. Where 
is the quadrupolar splitting for a deuteron attached to the ith carbon, e2qQ/h is the quadrupolar splitting constant (168 kHz for deuterons in C-2H bonds), and 
is the chain order parameter for a deuteron attached to the ith carbon of the acyl chain of POPC-d31. The order parameters calculated for the CD3 quadrupolar splitting should be multiplied by three according to procedures in the literature [46,47].
Figure 5 displays the smoothed segmental C-D bond order parameters (SCD) of the POPC-d31 acyl chains calculated by dePakeing the corresponding 2H NMR powder spectra shown in Figure 4. Figure 5 reveals a characteristic profile of decreasing order (SCD) with increasing distance from the glycerol backbone of POPC-d31 MLVs. Any decrease (if any) in the order parameter profile of POPC-d31-containing bilayers in the presence of (X) mol% of specific protein indicates more disorder and motion in the acyl chain region of the lipids when compared to the control sample (without protein). If the order parameter profile of POPC-d31-containing bilayers in the presence of (X) mol% protein is similar to the control sample, this indicates that this particular protein is not altering the order and motion in the acyl chain region of the lipids.
2.2. <sup>31</sup>P Solid-State NMR Spectroscopy of Phospholipid Head GroupsThe motionally averaged powder pattern spectra are characteristic of MLVs in the liquid crystalline phase (La) and are expected for phospholipids bilayers at a temperature well above the chain melting point transition temperature (Tm) [48]. Figure 6(a) shows the structure of the neutral phospholipid POPC (Tm = –2˚C) [39]. In addition, other phospholipids that includes 1-palmitoyl-2- oleoyl-sn-glycero-3-[phospho-L-serine] (POPS, Tm = 14 ˚C, Figure 6(b)), dioleoylphosphatidyl-glycerol (DOPG, Tm = –18˚C, Figure 6(c)) and dioleoylphosphatidylserine (DOPS, Tm = –11˚C, Figure 6(d)) structures are shown [39]. The static 31P NMR spectrum of each phospholipid head group shown in Figures 6(a)-(d) have a specific 31P chemical shift anisotropy (CSA; is equal s33-s11) width and lineshape. In several cases, the lineshapes of the static 31P NMR spectra and its corresponding CSA values have been successfully used to study the perturbation effect induced by drugs and proteins on phospholipids [11,14,49,50]. The addition of (X) mol% of specific protein to specific MLVs could alter the 31P CSA and line shape (Figure 7(b)) when compared to the control (Figure 7(a)) sample. This indicates that the protein of interest is interacting with the head group region of the MLVs.
2.3. <sup>13</sup>C Solid-State NMR Spectroscopy of Site-Specific <sup>13</sup>C-Labeled Peptides in MLVsThe secondary structure of 13C=O site-specific isotopically labeled peptides or proteins inserted into lipid bilayers can be probed utilizing 13C CPMAS solid-state NMR spectroscopy [15-18,50]. The local conformations of peptides and proteins can be characterized by examining the 13C=O chemical shifts of the 13C-labeled carbonyl of Ala, Leu, and Val. In general, the a-helical structure chemical shifts may vary within the data range (174 - 177 ppm) for different conformational-dependent changes (see Figure 8(a)) [17]. Also, a single 13C NMR peak at approximately 172.4 ppm can be attributed to either a b-sheet or an unstructured structural conformation of the peptide (see Figure 8(b)); whereas a single peak at approximately 176 ppm can be attributed to one a-helical structure conformation (see Figure 8(c)) [15].
2.4. <sup>2</sup>H Solid-State NMR Spectroscopy of Site-Specific <sup>2</sup>H-Labeled Peptides in MLVsAs mentioned previously, probing of how both segments of phosphorylated and unphosphorylated membrane proteins are moving within the phospholipid bilayers is crucial to describe the physiological functions. 2H solidstate NMR spectroscopy is a powerful well developed technique to study the structural and side-chain dynamic properties of membrane proteins in phospholipid bilayers [8,51-53]. The corresponding quadrupolar splitting and lineshapes of the 2H solid-state NMR spectra can be used to probe the molecular dynamics of the sidechain of selectively labeled residues in site-specific 2H-labeled integral membrane proteins [19-24].
In previous studies, methyl group motions have been well-characterized utilizing 2H NMR studies of CD3-labeled sites of alanines, valines and leucines [19,24,25,54-57]. For the isotopically labeled alanines (short aliphatic side-chains), the deuterated methyl group (CD3) rotates along the Ca-Cb bond and allows the deuterons to make jumps between three-sites described by a tertrahedral geometry (see Figure 9(a)) [58,59]. However, for Leu, the long aliphatic side-chain can be isotopically labeled at the δ- and/or ε-CD3 sites (see Figure 9 (b)) and the deuterium NMR powder pattern lineshapes will be strongly influenced by the motions about the Cg-Cd bond axis as well as by additional librational motion about the Ca-Cb and Cb-Cg bond axes at various temperatures [24,60].
It has been reported that if the CD3-methyl probe of a protein undergoes no motion other than those associated with the axial rotation about the C-CD3 bond in a randomly dispersed sample, the resultant 2H NMR spectra will consist of a Pake pattern with a 40 kHz quadrupolar splitting (see Figure 10(a)) [61]. However, residues located outside the membrane are expected to be more motionally averaged and yield an isotropic peak (see Figure 10(b)) [62].
2.5. <sup>15</sup>N Solid-State NMR Spectroscopy of Site Specific <sup>15</sup>N-Labeled Peptides in MLVs15N solid-state NMR spectroscopic studies are utilized to ascertain pertinent information on the backbone structure and dynamics of membrane proteins in phospholipid bilayers [25,26]. It provides important physiological and mechanistic information regarding the regulatory role of membrane proteins and it phosphorylated form in biological systems [64-66]. Generally, the immobile (without large amplitude motions) amide sites of specific 15N-labeled proteins yield a broad static 15N powder pattern (see Figure 11(a)); whereas, motionally averaged amide sites reveal isotropic peaks (see Figure 11(b)) [26].
2.6. <sup>15</sup>N Solid-State NMR Spectroscopy of Site-Specific <sup>15</sup>N-Labeled Peptides in Mechanically Oriented BilayersDetermining the structural topology of membrane proteins and its interaction with the lipids is critical to understand its physiological regulatory function. 15N solidstate NMR spectroscopy is a powerful tool to ascertain direct information regarding the structural topology of membrane proteins in oriented phospholipid bilayers [2].
In this approach, specific 15N labeled amide sites can be used to probe the alignment of the helix with respect to the bilayer normal. For example, this technique was used to probe the orientation of both the transmembrane and cytoplasmic domains of WT-PLB embedded inside mechanically oriented phospholipids [68]. Site-specific 15N-labeled WT-PLB were chosen at Ala11 (in the cytoplasmic domain) and Leu 42 and Leu 51 (both in the transmembrane helix) (see Figure 12).
A resonance peak at approximately 70 ppm (close to the s^ component of the chemical shift tensor of the corresponding powder spectrum, see Figure 13), indicates that this residue is oriented approximately perpendicular to the bilayer normal (transmembrane helix as shown in Figure 12) [2]. Also, a 15N resonance peak at approximately 210 ppm (close to the sêê component of the chemical shift tensor of the corresponding powder spectra, see Figure 13) indicates that the amide backbone vector of this residue is nearly parallel to the bilayer normal (the helix lies on the surface of the phospholipids bilayers as shown for cytoplasmic domain of the proposed structural model of WT-PLB presented in Figure 12 [2].