Characterization of the mechanism of interaction between α₁‐acid glycoprotein and lipid membranes by vacuum‐ultraviolet circular‐dichroism spectroscopy

Abstract

α 1 -Acid glycoprotein (AGP) interacts with lipid membranes as a peripheral membrane protein so as to decrease the drug-binding capacity accompanying the β!α conformational change that is considered a protein-mediated uptake mechanism for releasing drugs into membranes or cells. This study characterized the mechanism of interaction between AGP and lipid membranes by measuring the vacuum-ultraviolet circular-dichroism (VUVCD) spectra of AGP down to 170 nm using synchrotron radiation in the presence of five types of liposomes whose constituent phospholipid molecules have different molecular characteristics in the head groups (e.g., different net charges). The VUVCD analysis showed that the α-helix and β-strand contents and the numbers of segments of AGP varied with the constituent phospholipid molecules of liposomes, while combining VUVCD data with a neural-network method predicted that these membrane-bound conformations comprised several common long helix and small strand segments. The amino-acid composition of each helical segment of the conformations indicated that amphiphilic and positively charged helices formed at the N-and C-terminal regions of AGP, respectively, were candidate sites for the membrane interaction. The addition of 1 M sodium chloride shortened the C-terminal helix while having no effect on the length of the N-terminal one. These results suggest that the N-and C-terminal helices can interact with the membrane via hydrophobic and electrostatic interactions, respectively, demonstrating that the liposome-dependent conformations of AGP analyzed using VUVCD spectroscopy provide useful information for characterizing the mechanism of interaction between AGP and lipid membranes. K E Y W O R D Sα 1 -acid glycoprotein, electrostatic and hydrophobic interactions, membrane-bound conformation, secondary structure of protein, synchrotron radiation circular dichroism

Conformation of myelin basic protein bound to phosphatidylinositol membrane characterized by vacuum‐ultraviolet circular‐dichroism spectroscopy and molecular‐dynamics simulations

“…The MBP concentration was carefully determined by analyzing amino acids and measuring absorptions (V‐560, Jasco, JPN) (the molar extinction coefficient at 276 nm was 1.73 × 10 5 M −1 cm −1 ). A mildly acidic state (A‐state, pH 4.5) was used as the environment for the membrane interactions of MBP in this study, because protein‐membrane interactions are increased at mildly acidic pH without the presence of aggregation or turbidity 18,23,35‐38 …”

“…The turns and unordered structures estimated by the SELCON3 program were classified as “other structures” in the VUVCD‐NN method. The predictive accuracy of this method for the positions of α‐helix and β‐strand segments was 74.9% for 30 reference soluble proteins 26 and 73% for 15 integral membrane proteins, suggesting that this combination technique will be valuable for predicting the sequences of secondary structures of soluble proteins in the presence of liposomes 23,27,28 …”

“…CD spectroscopy is one of the most widely used techniques for monitoring protein structures in solution. Although CD does not reveal protein structure at the atomic resolution that is possible in X‐ray crystallography and NMR spectroscopy, it can be applied to the conformational analysis of both integral and nonintegral (or peripheral) membrane proteins 21‐23 . Further, vacuum‐ultraviolet circular‐dichroism (VUVCD) spectroscopy using synchrotron radiation has extended the protein CD spectrum into the vacuum‐ultraviolet region (down to 160 nm), 24,25 which allowed us to improve the accuracy of estimating the contents and numbers of α‐helix and β‐strand segments of proteins compared to conventional CD spectroscopy 21,26 and also to determine the positions of these segments on the amino‐acid sequence in combination with a neural‐network (NN) algorithm (the VUVCD‐NN method) 26 .…”

“…Although CD does not reveal protein structure at the atomic resolution that is possible in Xray crystallography and NMR spectroscopy, it can be applied to the conformational analysis of both integral and nonintegral (or peripheral) membrane proteins. [21][22][23] Further, vacuum-ultraviolet circular-dichroism (VUVCD) spectroscopy using synchrotron radiation has extended the protein CD spectrum into the vacuum-ultraviolet region (down to 160 nm), 24,25 which allowed us to improve the accuracy of estimating the contents and numbers of α-helix and β-strand segments of proteins compared to conventional CD spectroscopy 21,26 and also to determine the positions of these segments on the aminoacid sequence in combination with a neural-network (NN) algorithm (the VUVCD-NN method). 26 VUVCD analysis has been used to successfully estimate the contents, segments, positions, and orientations of secondary structures of various membrane-bound proteins (eg, α 1acid glycoprotein, β-amyloid protein, and β-lactoglobulin), 23,27,28 which indicates that this technique could be a powerful tool for characterizing the conformation of MBP in the presence of membrane.…”

Secondary Structure of Human De Novo Evolved Gene Product NCYM Analyzed by Vacuum-Ultraviolet Circular Dichroism

“…The method has been used for the structural analysis of unknown proteins in the native and other states so far ( 22 , 23 ).…”