A revolutionary way to observe cell trafficking

Membrane proteins are key targets for many drugs
.
They are located between
the inside and outside of the cell.
Some of them are called "transporters" and move certain substances into or out of the cellular environment
.
However, extracting and storing them for observation is particularly complex
.
A team from the University of Geneva (UNIGE), in collaboration with the University of Zurich (UZH), has developed an innovative way to study their structure in their native environment: cells
.
The technique is based on electron spin resonance spectroscopy
.
The results, just published in the journal Science Advances, could lead to the development of new drugs in the future
.

In living organisms, each cell is surrounded
by a cell membrane (or "cytoplasmic membrane").
This membrane consists of a double layer of
lipids.
It separates the contents of the cell from the immediate environment and regulates what enters
or leaves the cell.
Proteins attached to membranes are called "membrane proteins"
.
Located at the interface inside and outside the cell, they carry a variety of substances across the cell membrane – into or out of the cell – and play a vital role in cell signaling, i.
e.
in the cell's communication system, allowing them to coordinate metabolic processes, development and organization
.
As a result, membrane proteins account for more than
60% of current drug targets.

Difficult to study objects

Therefore, biophysical studies of their structure (the spatial organization that makes up amino acids) are necessary
.
To characterize them, scientists must extract these proteins from the cell membrane in which they reside and separate
them from all other proteins.
Once membrane proteins have been extracted, they cannot be studied
in aqueous solutions.
They must be kept in a
liquid solution consisting of detergents.
They can also be inserted into artificial membrane "nanodisks" made of proteins and lipids, or into
a pure lipid film.

In any case, these strategies remove them from their physiological environment, not allowing fine observation of their function
in situ.
Proteins that are detached from their natural environment may exhibit different structural properties that can mislead drug development
.

A revolutionary approach

The team, led by Professor Enrica Bordignon from the Department of Physical Chemistry at UNIGE's School of Science, in collaboration with Associate Professor Markus A.
Seeger of the UZH Institute of Medical Microbiology, developed a new method to study the role of membrane proteins in living cells; More precisely, on the
inner cell membrane of E.
coli.
To achieve this, the research team relied on a specific "tool": nanobodies
.

Enrica Bordignon explains: "These antibody fragments are able to recognize and bind to specific targets in a very efficient way, such as antigens or, in our case, membrane transporters
.
" Therefore, scientists artificially fabricate specific nanobodies for membrane transporters and use them to report their structures
directly.
: After insertion of E.
coli cells, two nanobodies target the desired membrane protein on the inner membrane of the cell and attach to
it.

New targets for certain drugs

Before that, a small magnetic probe (a molecule carrying unpaired electrons) was attached to
each nanobody.
Enrica Bordignon explains: "When two nanobodies bind to a transporter, we can measure the distance
between two magnetic probes in the cell using the EPR method.
" This technique is called "electron paramagnetic resonance spectroscopy" (EPR) or "electron spin resonance.
"
The measured distance is in the nanometer range (millionths of a millimeter).

Enrica Bordignon enthuses: "This is the first time we have succeeded in obtaining a clear picture of the conformation of a membrane protein in a real environment, and we can track the changes
caused when we modify a single amino acid to another.
"

"The development of this new strategy is the result of
our excellent and challenging teamwork between our two groups, UNIGE and UZH.
In particular, the persistence of the two first authors, Dr.
Laura Galazzo (UNIGE) and Dr.
Gianmarco Meier (UZH), made this project a success after five years of research", stresses the researchers
.

This new strategy allows precise determination of the properties of
membrane proteins in the immediate environment.
It offers the possibility of better understanding how these proteins transport certain substances in and out of
cells.
This method also has the advantage of
easily transposing mammalian cells.
It can be used to better understand and better target membrane proteins that reject certain anticancer drugs outside the cell, thereby combating multidrug resistance
.

Laura Galazzo, Gianmarco Meier, Dovile Januliene, Kristian Parey, Dario De Vecchis, Bianca Striednig, Hubert Hilbi, Lars V.
Schä fer, Ilya Kuprov, Arne Moeller, Enrica Bordignon, Markus A.
Seeger.
The ABC transporter MsbA adopts the wide inward-open conformation in E.
coli cells.
Science Advances, 2022; 8 (41)