Structural changes in a binary mixed phospholipid bilayer of DOPG and DOPS upon saposin C interaction at acidic pH utilizing 31P and 2H solid-state NMR spectroscopy

Abstract

Saposin C (Sap C) is known to stimulate the catalytic activity of the lysosomal enzyme glucosylceramidase (GCase) that facilitates the hydrolysis of glucosylceramide to ceramide and glucose. Both Sap C and acidic phospholipids are required for full activity of GCase. In order to better understand this interaction, mixed bilayer samples prepared from dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylserine (DOPS) (5:3 ratio) and Sap C were investigated using (2)H and (31)P solid-state NMR spectroscopy at temperatures ranging from 25 to 50 degrees C at pH 4.7. The Sap C concentrations used to carry out these experiments were 0 mol%, 1 mol% and 3 mol% with respect to the phospholipids. The molecular order parameters (S(CD)) were calculated from the dePaked (2)H solid-state NMR spectra of Distearoyl-d70-phosphatidylglycerol (DSPG-d70) incorporated with DOPG and DOPS binary mixed bilayers. The S(CD) profiles indicate that the addition of Sap C to the negatively charged phospholipids is concentration dependent. S(CD) profiles of 1 mol% of the Sap C protein show only a very slight decrease in the acyl chain order. However, the S(CD) profiles of the 3 mol% of Sap C protein indicate that the interaction is predominantly increasing the disorder in the first half of the acyl chain near the head group (C1-C8) indicating that the amino and the carboxyl termini of Sap C are not inserting deep into the DOPG and DOPS mixed bilayers. The (31)P solid-state NMR spectra show that the chemical shift anisotropy (CSA) for both phospholipids decrease and the spectral broadening increases upon addition of Sap C to the mixed bilayers. The data indicate that Sap C interacts similarly with the head groups of both acidic phospholipids and that Sap C has no preference to DOPS over DOPG. Moreover, our solid-state NMR spectroscopic data agree with the structural model previously proposed in the literature [X. Qi, G.A. Grabowski, Differential membrane interactions of saposins A and C. Implication for the functional specificity, J. Biol. Chem. 276 (2001) 27010-27017] [1].

Phospholamban and Its Phosphorylated Form Interact Differently with Lipid Bilayers: A31P,2H, and13C Solid-State NMR Spectroscopic Study

“…Then, the quadrupolar splittings were directly measured from the dePaked spectra and converted into order parameters as described before (26,27). For numbering, the 2 H nuclei attached to the terminal methyl carbons were assigned carbon number 15.…”

Solid-State NMR Spectroscopic Approaches to Investigate Dynamics, Secondary Structure and Topology of Membrane Proteins

“…and lineshape. In several cases, the lineshapes of the static 31 P 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 31 P CSA and line shape (Figure 7(b)) when compared to the control (Figure 7(a)) sample.…”

“…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 CD 3 methyl groups (see Figure 1) at the end of the acyl chains are the smallest and close to 0 kHz because he fastest frequency.…”

“…From the perspective of the perpetuated lipids, 2 H 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][9][10][11][12][13]. Moreover, 31 P solid-state NMR spectroscopy can be used to investigate the interaction of peptides, proteins and drugs with phospholipid head groups [11][12][13][14].…”

“…From the perspective of the perpetuated lipids, 2 H 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][9][10][11][12][13]. Moreover, 31 P solid-state NMR spectroscopy can be used to investigate the interaction of peptides, proteins and drugs with phospholipid head groups [11][12][13][14]. The secondary structure of 13 C=O site-specific isotopically labeled peptides or proteins inserted into lipid bilayers can be probed utilizing 13 C CPMAS solid-state NMR spectroscopy [15][16][17][18].…”