真空紫外円二色性によるタンパク質と生体膜の相互作用機構の解析

広島大学(Hiroshima University)博士(理学)Doctor of Sciencedoctora

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

α1-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.This work was financially supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (numbers 19K06587 and 15K07028).[This article is part of the Special Issue: In honor and memory of Prof. Koji Nakanishi. See the first articles for this special issue previously published in Volumes 31:12, 32:3, and 32:4. More special articles will be found in this issue as well as in those to come.

Formation of β-Strand Oligomers of Antimicrobial Peptide Magainin 2 Contributes to Disruption of Phospholipid Membrane

The antimicrobial peptide magainin 2 (M2) interacts with and induces structural damage in bacterial cell membranes. Although extensive biophysical studies have revealed the interaction mechanism between M2 and membranes, the mechanism of membrane-mediated oligomerization of M2 is controversial. Here, we measured the synchrotron-radiation circular dichroism and linear dichroism (LD) spectra of M2 in dipalmitoyl-phosphatidylglycerol lipid membranes in lipid-to-peptide (L/P) molar ratios from 0–26 to characterize the conformation and orientation of M2 on the membrane. The results showed that M2 changed from random coil to α-helix structures via an intermediate state with increasing L/P ratio. Singular value decomposition analysis supported the presence of the intermediate state, and global fitting analysis revealed that M2 monomers with an α-helix structure assembled and transformed into M2 oligomers with a β-strand-rich structure in the intermediate state. In addition, LD spectra showed the presence of β-strand structures in the intermediate state, disclosing their orientations on the membrane surface. Furthermore, fluorescence spectroscopy showed that the formation of β-strand oligomers destabilized the membrane structure and induced the leakage of calcein molecules entrapped in the membrane. These results suggest that the formation of β-strand oligomers of M2 plays a crucial role in the disruption of the cell membrane

Heat-Induced Conformational Transition Mechanism of Heat Shock Factor 1 Investigated by Tryptophan Probe

A transcriptional regulatory system called heat shock response (HSR) has been developed in eukaryotic cells to maintain proteome homeostasis under various stresses. Heat shock factor-1 (Hsf1) plays a central role in HSR, mainly by upregulating molecular chaperones as a transcription factor. Hsf1 forms a complex with chaperones and exists as a monomer in the resting state under normal conditions. However, upon heat shock, Hsf1 is activated by oligomerization. Thus, oligomerization of Hsf1 is considered an important step in HSR. However, the lack of information about Hsf1 monomer structure in the resting state, as well as the structural change via oligomerization at heat response, impeded the understanding of the thermosensing mechanism through oligomerization. In this study, we applied solution biophysical methods, including fluorescence spectroscopy, nuclear magnetic resonance, and circular dichroism spectroscopy, to investigate the heat-induced conformational transition mechanism of Hsf1 leading to oligomerization. Our study showed that Hsf1 forms an inactive closed conformation mediated by intramolecular contact between leucine zippers (LZs), in which the intermolecular contact between the LZs for oligomerization is prevented. As the temperature increases, Hsf1 changes to an open conformation, where the intramolecular LZ interaction is dissolved so that the LZs can form intermolecular contacts to form oligomers in the active form. Furthermore, since the interaction sites with molecular chaperones and nuclear transporters are also expected to be exposed in the open conformation, the conformational change to the open state can lead to understanding the regulation of Hsf1-mediated stress response through interaction with multiple cellular components