Researchers have been elucidating the structures of complex molecules for over 100 years, using a variety of techniques. Some molecular structures, however, are more difficult to solve than others.
Transmembrane proteins are massive molecules that span the lipid (fat) bilayers of the cell membrane to perform a variety of important functions, like controlling the flow of ions (charged atoms) into and out of the cell, detecting extracellular molecules, joining two cells together and accelerating chemical reactions. Seven transmembrane (7TM) proteins, in particular, are large molecules, also known as G-protein coupled receptors, that bind to extracellular molecules and activate a multitude of different responses inside the cell. Like their name suggests, these proteins have seven transmembrane regions that span the lipid bilayer of the cell membrane.
X-ray crystallography and cryo-electron microscopy (EM) have been used to determine high-level structures of 7TM proteins, but determining the high-resolution, native structure of 7TM proteins in the lipid bilayer has been challenging. High-resolution structural information for molecules is typically generated using nuclear magnetic resonance (NMR) spectroscopy, a technique that uses a strong magnetic field and radio waves to determine the structure of molecules.
To address this issue, a group of researchers from YOKOHAMA National University, Bruker Biospin, Bruker Japan, and The University of Tokyo developed a method to determine the high-resolution structure of schizorhodopsin SzR1, at 7TM protein proton pump. Importantly, these results were obtained in a fully protonated form of SzR1, where all amino acids that can accept a proton (H+) are filled, in a lipid bilayer, allowing the protein to maintain its native, solid state.
The team published their paper, "Ultrafast 160 kHz MAS enables high-resolution 1H-detected solid-state NMR of a fully protonated seven-transmembrane protein," on Aug. 11 in the journal Chemical Communications .
"Membrane proteins are biologically important targets involved in cellular signaling and molecular transport, and their functions are closely linked to their structure and dynamics in the membrane environment. However, strong 1H–1H [proton–proton] dipolar interactions broaden NMR signals, making it difficult to obtain detailed structural information without deuteration. We therefore aimed to determine whether ultrafast magic angle spinning (MAS) could overcome this limitation and enable high-resolution 1H-detected solid-state NMR of fully protonated membrane proteins in a native-like lipid environment," said Izuru Kawamura, professor in the Graduate School of Engineering Science at YOKOHAMA National University and senior author of the research study.
Deuteration is a process where researchers swap normal hydrogen atoms (1H) with deuterium atoms (2H), a heavier and more stable isotope of hydrogen. While this process can help with some of the signal problems associated with proton–proton dipolar interactions, deuteration is expensive, can cause changes in protein structure and can cause other signal-degradation issues.
Instead, the team used ultrafast magic angle spinning (MAS) NMR to determine the structure of the 7TM protein SzR1. MAS spins the sample at much higher speeds than normal NMR, in this case 160 kHz (or 160,000 cycles per second), with the sample oriented 54.74° relative to the magnetic field. MAS tends to cancel out any directional forces that occur on the protein over time, sharpening the NMR spectra and structural data.
"The key message is that ultrafast MAS at 160 kHz using a 0.4 mm rotor enables high-resolution 1H-detected solid-state NMR of a fully protonated seven-transmembrane protein in a hydrated lipid bilayer using only a sub-milligram amount of protein. We achieved substantially narrower 1H linewidths and could clearly detect signals from the protonated [SzR1]. This demonstrates that ultrafast MAS can provide detailed structural information while preserving the native-like membrane protein [structure]," said Kawamura.
Despite this advance in solid-state NMR (ssNMR) technique, the team isn't finished optimizing 7TM protein structure measurements.
"The next step is to combine ultrafast MAS with ultrahigh magnetic fields above 1 GHz to further improve sensitivity, spectral resolution and chemical shift dispersion. Ultimately, we aim to establish 1H-detected solid-state NMR with ultrafast MAS as a powerful approach for investigating the structure and dynamics of membrane proteins in native-like lipid environments, including pharmacologically important targets such as G-protein coupled receptors and membrane transporters," said Kawamura.
Haruto Nakajima and Takumi Kanazawa from the Graduate School of Engineering Science at YOKOHAMA National University in Yokohama, Japan; Kristof Grohe and Sebastian Wegner from Bruker Biospin in Ettlingen, Germany; Hideaki Kimura from Biospin Japan in Yokohama, Japan; and Keiichi Inoue from the Graduate School of Frontier Sciences and The Institute for Solid-State Physics at The University of Tokyo in Tokyo and Kashiwa, Japan, respectively, also contributed to this research.
This work was supported by by JSPS KAKENHI Grants-in-Aid (Grant Number: JP21H05229, JP23K18090, JP24H02268, and JP25H00424), JST CREST (Grant Number: JPMJCR21B2, JPMJCR22N2), JST SPRING (Grant Number: JPMJSP2178), MEXT Promotion of Development of a Joint Usage/Research System Project: Coalition of Universities for Research Excellence Program (CURE) (Grant Number: JPMXP1323015482) and NMR Platform Program of Japan NF23-01-0161 and PF25-01-091 (Grant Number: JPMXS0450100021).
