The human body contains a vast number of proteins that are collectively called the proteome. Around 80,000 to 400,000 proteins circulate in the human cells, organs, and tissues, performing a wide range of functions crucial for life. When these proteins go awry, they lead to a range of serious diseases.
Wei Liu is a researcher in the Biodesign Center for Applied Structural Discovery and ASU’s School of Molecular Sciences. Image Credit: Arizona State University.
Scientists from the Biodesign Center for Applied Structural Discovery and the School of Molecular Sciences at Arizona State University, and their collaborators have now explored a crucially significant major class of proteins, which decorate the external membranes of cells. Membrane proteins like these usually serve as receptors for binding molecules, triggering signals that can modify the behavior of cells in many different ways.
A new method to achieve structural data of membrane proteins in explicit detail has been explained in the recent study. This approach involves the use of cryogenic electron microscopy (or cryo-EM) techniques, which are an innovative suite of tools.
Moreover, the use of so-called Microcrystal electron diffraction (MicroED) and LCP crystallization helps reveal the proteins’ structural details that have been mostly inaccessible through standard techniques, like X-ray crystallography.
The results describe the initial use of LCP-embedded microcrystals to expose the high-resolution structural details of proteins through MicroED. The study featured on the cover of the latest issue of the Cell Press journal, Structure.
LCP was a great success in membrane protein crystallization. The new extensive application of LCP-MicroED offers promise for improved approaches for structural determination from challenging protein targets. These structural blueprints can be used to facilitate new therapeutic drug design from more precise insights.”
Wei Liu, Study Corresponding Author, Arizona State University
G-protein-coupled receptors (GPCRs) are a group of membrane proteins that hold a specific interest. They represent the most varied and largest group of membrane receptors found in eukaryotic organisms, such as humans.
The physiological activities of GPCRs are so crucial that they are an important target for a variety of therapeutic drugs. But this is where issues emerge, because establishing the comprehensive structure of membrane proteins—a vital precursor to precise drug design— poses immense challenges.
The X-ray crystallography technique has been used for studying the atomic-scale structures and also the dynamic behavior of several proteins. Here, crystallized protein samples being studied are bombarded with an X-ray beam, causing diffraction patterns, which emerge on a screen. Arranging scores of diffraction pictures helps assemble a high-resolution 3D structural image using computers.
Yet a majority of the membrane proteins, such as GPCRs, do not form huge, well-ordered crystals suitable for X-ray crystallography. Such proteins are also fragile and easily impaired by X-rays.
To solve the problem, unique devices called X-ray free electron lasers (XFELS) had to be used. Such devices can provide a brilliant burst of X-ray light that lasts just femtoseconds, (a femtosecond is equivalent to one quadrillionth of a second or approximately the time taken by a light ray to traverse the diameter of a virus). The method of serial femtosecond X-ray crystallography enables scientists to acquire a refraction image before the crystalized sample is damaged.
Nonetheless, crystallization of several membrane proteins continues to be a very difficult and inaccurate art and only a few of these gargantuan XFEL machines are available in the world.
Enter MicroED and cryogenic electron microscopy. This innovative method involves flash-freezing protein crystals in a thin coating of ice, subsequently exposing them to an electron beam. As in the case of X-ray crystallography, this technique makes use of diffraction patterns, but this time from electrons instead of X-rays, to organize final detailed structures.
The MicroED method excels in collecting information from crystals that are too tiny and irregular to be utilized in traditional X-ray crystallography.
In the latest research, scientists employed two sophisticated methods simultaneously to create high-resolution diffraction images of two significant model proteins—the A2A adenosine receptor and Proteinase K, whose roles include cardiac vasodilation, modulation of neurotransmitters in the brain, cardiac vasodilation and T-cell immune response.
The proteins were implanted in a unique type of crystal called LCP or a lipidic cubic phase crystal, which imitates the native environment where such proteins exist naturally.The LCP samples were subsequently subjected to electron microscopy, with the help of the MicroED method, which helps image very thin, sub-micron-sized crystals.
Moreover, when LCP crystals are continuously rotated under the electron microscope, multiple diffraction patterns can be obtained from a single crystal with a very low, damage-free electron dose.
The potential to analyze proteins that can only form nano- or microcrystals pave the way to the structural determination of several crucially significant membrane proteins that have escaped traditional means of analysis, especially GPCRs.
Zhu, L., et al. (2020) Structure Determination from Lipidic Cubic Phase Embedded Microcrystals by MicroED. Structure. doi.org/10.1016/j.str.2020.07.006.