New techniques probe vital and elusive proteins

Date:
October 6, 2020
Source:
Arizona State University
Summary:
Researchers have investigated a critically important class of
proteins, which adorn the outer membranes of cells. Such membrane
proteins often act as receptors for binding molecules, initiating
signals that can alter cell behavior in a variety of ways.



FULL STORY ==========================================================================
The number of proteins in the human body, collectively known as the
proteome, is vast. Somewhere between 80,000 and 400,000 proteins circulate
in our cells, tissues and organs, carrying out a broad range of duties essential for life.

When proteins go awry, they are responsible for a myriad of serious
diseases.


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Now, researchers at the Biodesign Center for Applied Structural Discovery
and ASU's School of Molecular Sciences, along with their colleagues, investigate a critically important class of proteins, which adorn the
outer membranes of cells. Such membrane proteins often act as receptors
for binding molecules, initiating signals that can alter cell behavior
in a variety of ways.

A new approach to acquiring structural data of membrane proteins in
startling detail is described in the new study. Cryogenic electron
microscopy (or cryo- EM) methods, a groundbreaking suite of tools, is
used. Further, use of so- called LCP crystallization and Microcrystal
electron diffraction (MicroED) help unveil structural details of proteins
that have been largely inaccessible through conventional approaches like
X-ray crystallography.

The findings describe the first use of LCP-embedded microcrystals to
reveal high-resolution protein structural details using MicroED. The
new research graces the cover of the current issue of the Cell Press
journal Structure.

"LCP was a great success in membrane protein crystallization, according
to Wei Liu, a corresponding author of the new study. "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." One class of membrane proteins of particular interest are the G-protein-coupled receptors (GPCRs), which
form the largest and most varied group of membrane receptors found in eukaryotic organisms, including humans.



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The physiological activities of GPCRs are so important that they
are a major target for a wide range of therapeutic drugs. This is
where problems arise however, as determining the detailed structure of
membrane proteins -- an essential precursor to accurate drug design --
often poses enormous challenges.

The technique of X-ray crystallography has been used to investigate the
atomic- scale structures and even dynamic behavior of many proteins. Here, crystallized samples of the protein under study are struck with an X-ray
beam, causing diffraction patterns, which appear on a screen. Assembling thousands of diffraction snapshots allows a high-resolution 3D structural
image to be assembled with the aid of computers.

Yet many membrane proteins, including GPCRs, don't form large,
well-ordered crystals appropriate for X-ray crystallography. Further,
such proteins are delicate and easily damaged by X-radiation. Getting
around the problem has required the use of special devices known as X-ray
free electron lasers or XFELS, which can deliver a brilliant burst of
X-ray light lasting mere femtoseconds, (a femtosecond is equal to one quadrillionth of a second or about the time it takes a light ray to
traverse the diamere of a virus). The technique of serial femtosecond
X-ray crystallography allows researchers to obtain a refraction image
before the crystalized sample is destroyed.

Nevertheless, crystallization of many membrane proteins remains an
extremely difficult and imprecise art and only a handful of these
gargantuan XFEL machines exist in the world.

Enter cryogenic electron microscopy and MicroED. This ground-breaking
technique involves flash-freezing protein crystals in a thin veneer
of ice, then subjecting them to a beam of electrons. As in the case of
X-ray crystallography, the method uses diffraction patterns, this time
from electrons rather than X-rays, to assemble final detailed structures.

MicroED excels in collecting data from crystals too small and irregular
to be used for conventional X-ray crystallography. In the new study, researchers used two advanced techniques in tandem in order to produce high-resolution diffraction images of two important model proteins:
Proteinase K and the A2A adenosine receptor, whose functions include
modulation of neurotransmitters in the brain, cardiac vasodilation and
T-cell immune response.

The proteins were embedded in a special type of crystal known as a
lipidic cubic phase or LCP crystal, which mimics the native environment
such proteins naturally occur in. The LCP samples were then subjected
to electron microscopy, using the MicroED method, which permits
the imaging of extremely thin, sub- micron-sized crystals. Further,
continuous rotation of LCP crystals under the electron microscope allows multiple diffraction patterns to be acquired from a single crystal with
an extremely low, damage-free electron dose.

The ability to examine proteins that can only form micro- or nanocrystals
opens the door to the structural determination of many vitally important membrane proteins that have eluded conventional means of investigation, particularly GPCRs.


========================================================================== Story Source: Materials provided by Arizona_State_University. Original
written by Richard Harth. Note: Content may be edited for style and
length.


========================================================================== Journal Reference:
1. Lan Zhu, Guanhong Bu, Liang Jing, Dan Shi, Ming-Yue Lee, Tamir
Gonen, Wei
Liu, Brent L. Nannenga. Structure Determination from Lipidic Cubic
Phase Embedded Microcrystals by MicroED. Structure, 2020; 28 (10):
1149 DOI: 10.1016/j.str.2020.07.006 ==========================================================================

Link to news story: https://www.sciencedaily.com/releases/2020/10/201006114300.htm

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