{"title":"Dynamic Observation of the Membrane Interaction Processes of β-Lactoglobulin by Time-Resolved Vacuum-Ultraviolet Circular Dichroism","authors":"Satoshi Hashimoto, and , Koichi Matsuo*, ","doi":"10.1021/acs.analchem.4c00556","DOIUrl":null,"url":null,"abstract":"<p >The elucidation of protein–membrane interactions is pivotal for comprehending the mechanisms underlying diverse biological phenomena and membrane-related diseases. In this investigation, vacuum-ultraviolet circular dichroism (VUVCD) spectroscopy, utilizing synchrotron radiation (SR), was employed to dynamically observe membrane interaction processes involving water-soluble proteins at the secondary-structure level. The study utilized a time-resolved (TR) T-shaped microfluidic cell, facilitating the rapid and efficient mixing of protein and membrane solutions. This system was instrumental in acquiring measurements of the time-resolved circular dichroism (TRCD) spectra of β-lactoglobulin (bLG) during its interaction with lysoDMPG micelles. The results indicate that bLG undergoes a β–α conformation change, leading to the formation of the membrane-interacting state (M-state), with structural alterations occurring in more than two steps. Global fitting analysis, employing biexponential functions with all of the TRCD spectral data sets, yielded two distinct rate constants (0.18 ± 0.01 and 0.06 ± 0.003/s) and revealed a unique spectrum corresponding to an intermediate state (I-state). Secondary-structure analysis of bLG in its native (N-, I-, and M-states) highlighted that structural changes from the N- to I-states predominantly occurred in the N- and C-terminal regions, which were prominently exposed to the membrane. Meanwhile, transitions from the I- to M-states extended into the inner barrel regions of bLG. Further examination of the physical properties of α-helical segments, such as effective charge and hydrophobicity, revealed that the N- to I- and I- to M-state transitions, which are ascribed to first- and second-rate constants, respectively, are primarily driven by electrostatic and hydrophobic interactions, respectively. These findings underscore the capability of the TR-VUVCD system as a robust tool for characterizing protein–membrane interactions at the molecular level.</p>","PeriodicalId":27,"journal":{"name":"Analytical Chemistry","volume":null,"pages":null},"PeriodicalIF":6.7000,"publicationDate":"2024-06-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Analytical Chemistry","FirstCategoryId":"92","ListUrlMain":"https://pubs.acs.org/doi/10.1021/acs.analchem.4c00556","RegionNum":1,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"CHEMISTRY, ANALYTICAL","Score":null,"Total":0}
引用次数: 0
Abstract
The elucidation of protein–membrane interactions is pivotal for comprehending the mechanisms underlying diverse biological phenomena and membrane-related diseases. In this investigation, vacuum-ultraviolet circular dichroism (VUVCD) spectroscopy, utilizing synchrotron radiation (SR), was employed to dynamically observe membrane interaction processes involving water-soluble proteins at the secondary-structure level. The study utilized a time-resolved (TR) T-shaped microfluidic cell, facilitating the rapid and efficient mixing of protein and membrane solutions. This system was instrumental in acquiring measurements of the time-resolved circular dichroism (TRCD) spectra of β-lactoglobulin (bLG) during its interaction with lysoDMPG micelles. The results indicate that bLG undergoes a β–α conformation change, leading to the formation of the membrane-interacting state (M-state), with structural alterations occurring in more than two steps. Global fitting analysis, employing biexponential functions with all of the TRCD spectral data sets, yielded two distinct rate constants (0.18 ± 0.01 and 0.06 ± 0.003/s) and revealed a unique spectrum corresponding to an intermediate state (I-state). Secondary-structure analysis of bLG in its native (N-, I-, and M-states) highlighted that structural changes from the N- to I-states predominantly occurred in the N- and C-terminal regions, which were prominently exposed to the membrane. Meanwhile, transitions from the I- to M-states extended into the inner barrel regions of bLG. Further examination of the physical properties of α-helical segments, such as effective charge and hydrophobicity, revealed that the N- to I- and I- to M-state transitions, which are ascribed to first- and second-rate constants, respectively, are primarily driven by electrostatic and hydrophobic interactions, respectively. These findings underscore the capability of the TR-VUVCD system as a robust tool for characterizing protein–membrane interactions at the molecular level.
期刊介绍:
Analytical Chemistry, a peer-reviewed research journal, focuses on disseminating new and original knowledge across all branches of analytical chemistry. Fundamental articles may explore general principles of chemical measurement science and need not directly address existing or potential analytical methodology. They can be entirely theoretical or report experimental results. Contributions may cover various phases of analytical operations, including sampling, bioanalysis, electrochemistry, mass spectrometry, microscale and nanoscale systems, environmental analysis, separations, spectroscopy, chemical reactions and selectivity, instrumentation, imaging, surface analysis, and data processing. Papers discussing known analytical methods should present a significant, original application of the method, a notable improvement, or results on an important analyte.