Solid-state NMR spectroscopy is routinely used to determine the structural and dynamic properties of both membrane proteins and peptides in phospholipid bilayers [1-26]. From the perspective of the perpetuated lipids, 2H solid-state NMR spectroscopy can be used to probe the effect of embedded proteins on the order and dynamics of the acyl chains of phospholipid bilayers [8-13]. Moreover, 31P solid-state NMR spectroscopy can be used to investigate the interaction of peptides, proteins and drugs with phospholipid head groups [11-14]. The 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]. Also, solid-state NMR spectroscopic studies can be utilized to ascertain pertinent informa- tion on the backbone and side-chain dynamics of 2H- and 15N-labeled proteins, respectively, in phospholipid bilayers [19-26]. Finally, specific 15N-labeled amide sites on a protein embedded inside oriented bilayers can be used to probe the alignment of the helices with respect to the bilayer normal [2]. A brief summary of all these solid-state NMR ap- proaches are provided in this minireview.
Membrane proteins make up approximately one-third of the total number of known proteins [
Multilamellar 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
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 [
In
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
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].
The 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) [
The 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
As 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
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
15N 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
Determining 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 [
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 [
A resonance peak at approximately 70 ppm (close to the s^ component of the chemical shift tensor of the corresponding powder spectrum, see
Solid-state NMR spectroscopy is routinely used to determine the structural and dynamic properties of both membrane proteins and peptides in phospholipid bilayers. Together, 2H, 31P, 13C and 15N solid-state NMR spectroscopy can be used to probe the effect of embedded proteins on the order and dynamics of the acyl chains, phospholipid head group as well as the secondary structure of site-specific isotopically labeled amino acid and helix orientation with respect to the membrane. A summary of all these solid-state NMR approaches are provided in this mini-review.