Hiroshima University succeeds at visualizing changes in protein structures during interactions with biomembranes: Expectations for clarification of complex life phenomena

Graduate Student Satoshi Hashimoto of the Graduate School of Advanced Science and Engineering, and Associate Professor Koichi Matsuo of the Research Institute for Synchrotron Radiation Science at Hiroshima University, have announced that they have developed a microfluidic device capable of efficiently mixing low-volume protein and biomembrane solutions. They installed this device in a vacuum-ultraviolet circular dichroism spectrophotometer using synchrotron radiation to successfully visualize the dynamic structural changes of a protein during its interaction with biomembrane in a wide time range of milliseconds to minutes. Their systems are expected to contribute to the clarification of complex biological phenomena occurring on biomembranes. The results were published in the international journal Analytical Chemistry on June 22.

Biomembrane-binding proteins are involved in a wide variety of important biological phenomena, including Parkinson's disease and the cellular transport of drugs. Therefore, clarification of the mechanisms underlying these phenomena requires the visualization of the dynamic structural changes of proteins during their interaction with biomembranes.

Previously, the research group used vacuum-ultraviolet circular dichroism (VUVCD) spectroscopy to detect protein structures using wavelengths down to ranges that cannot be measured with commercially available instruments. Furthermore, the research group used a machine learning method (VUVCD-NN method) to predict which regions in the amino acid sequences form regular structures such as α-helix and β-strand structures for structural analysis of proteins. The proposed method was applicable only to static structural information before and after interaction with biomembranes.

In this study, the research group constructed a microfluidic device in which two different biological sample solutions (a protein solution and biomembrane solution) could be efficiently mixed and the device was installed in the VUVCD system. The observation of dynamic protein changes was examined. They attempted to clarify the mechanism of the interaction between the biomembrane and β-lactoglobulin (bLG), which transports compounds constituting the membrane.

The system developed in this study consists of syringe pumps to deliver solutions, a microfluidic device, an optical system including a condenser lens to minimize the size of synchrotron radiation, and a detector to measure the intensity of radiation.

The results confirmed that the obtained spectra can be used to predict the content and number of regular structures in each bLG structure, as well as their positions at the amino acid sequence level. The microfluidic device can be used not only for proteins and biomembranes, but also for various biomolecules, such as polysaccharides and nucleic acids, as well as exosomes and nanocapsules.

Matsuo said, "We steadily proceeded with the study, overcoming problems at each step starting with designing the small device, its evaluation, confirmation of the drive system, preparation of biological samples, measurement of spectra, and data analysis. This was the difficult part (on the contrary, the fun part). We believe that the visualization of interactions using the system constructed in this study will help us understand the molecular-level mechanisms of various life phenomena and diseases that have been important but have not been clarified and find broad applications and applied research areas."

Journal Information
Publication: Analytical Chemistry
Title: Dynamic Observation of the Membrane Interaction Processes of β-Lactoglobulin by Time-Resolved Vacuum-Ultraviolet Circular Dichroism
DOI: 10.1021/acs.analchem.4c00556