q&more
My watch list
my.chemie.de  
Login  

News

Observing proteins in their natural environment

© RUB, Marquard

Laura Galazzo (left) and Enrica Bordignon use tiny parts of antibodies to examine proteins in detail.

11-Feb-2020: Proteins can be responsible for the fact that the active ingredients of drugs are simply released from the target cells. You can watch them do this now.

Certain medications, such as those used to treat cancer, lose their effect because proteins in the membrane of the target cell simply expel them again. A team at Ruhr-Universität Bochum (RUB) was able to observe a responsible transport protein in its natural environment for the first time. They labelled it with small sequences of antibodies to which a contrast agent was linked. By detecting the spin of the metal contrast agent with electron paramagnetic resonance spectroscopy (EPR, in short), they could draw conclusions on the state of the protein. The team led by Professor Enrica Bordignon and Dr. Laura Galazzo from the cluster of excellence Ruhr Explores Solvation Resolv in collaboration with Professor Markus Seeger’s group from the University of Zurich reports on the method in the journal PNAS on 4 February 2020.

Finding and binding nanobodies to the protein

Until now, it has only been possible to examine membrane proteins in isolation, namely in detergent micelles or in-vitro-created membrane bilayers, which poses the risk that they will lose their properties and dynamics, which are crucial for their function. The team from Resolv was now able to observe a protein directly in its natural environment, namely in the membrane of Escherichia coli bacteria. “It is very cramped and crowded there,” describes Enrica Bordignon. The team’s trick is to use two sequences of antibodies measuring just a few nanometres in size, called nanobodies, as labels. “We use precisely those sequences that can recognise and bind to certain sections of the protein,” explains Laura Galazzo.

Markus Seeger’s team took care of selecting the right nanobodies. “Thanks to the selection platform we developed, which bypasses the immunisation of animals, any laboratory can quickly produce synthetic nanobodies for any purpose. This means a step towards their use in structural biology, as this work shows,” says Seeger.

How to make the nanobodies visible in EPR? Gadolinium ions are used as a contrast agent for magnetic resonance imaging and are detectable by EPR due to their electron spin, therefore, they were attached to two selected nanobodies. As the labelled nanobodies could not yet efficiently be inserted into the bacteria, the scientists used a biochemical trick: the E. coli cells were turned inside out so that the inside of the membrane was exposed to the outside. In this way the sections of the protein targeted by the nanobodies become accessible.

Signal only with a certain shape

“The nanobodies immediately bind to the specific sequence of the membrane protein that they recognise and cannot detach again,” says Enrica Bordignon. The cells treated in this way were then examined by the researchers using EPR. “We were only able to receive a signal when two nanobodies were within close proximity to each other,” explains Laura Galazzo. This was precisely the case when the protein assumes a conformation that expels active substances from the cell.

“Our work shows that we can successfully measure distances in the range of 1.5 to 6 nanometres in native membranes,” says Enrica Bordignon. “In the next step, we want to insert the nanobodies into bacteria to observe the membrane protein during action in living cells. “This technique opens up undreamt-of possibilities,” says Laura Galazzo.

Original publication:
Laura Galazzo, Gianmarco Meier, M. Hadi Timachi, Cedric A. J. Hutter, Markus A. Seeger, Enrica Bordignon; "Spin-labeled nanobodies as protein conformational reporters for electron paramagnetic resonance in cellular membranes"; PNAS; 2020.

Facts, background information, dossiers

  • proteins
  • transport proteins
  • membrane proteins
  • Escherichia coli
  • nanobodies
  • electron paramagnet…
  • EPR spectroscopy

More about Ruhr-Universität Bochum

  • News

    RNA structures by the thousands

    Every cell contains thousands of different RNA molecules. In the past, it would have taken many research careers to decipher their structures using traditional methods. But not anymore. Researchers from Bochum and Münster have developed a new method to determine the structures of all RNA mo ... more

    Water molecules dance in three

    Water may look like a simple liquid. However, it is anything but simple to analyse. The unique properties of water are only understandable, if scientists observe the interaction of the molecules. An international team of scientists lead by Professor Martina Havenith from Ruhr-Universität Bo ... more

    Artificial intelligence identifies optimal material formula

    Nanostructured layers boast countless potential properties – but how can the most suitable one be identified without any long-term experiments? A team from the Materials Discovery Department at Ruhr-Universität Bochum (RUB) has ventured a shortcut: using a machine learning algorithm, the re ... more

  • q&more articles

    Light plus current: The formula for researching what happens to individual nanoparticles

    A combination of dark-field microscopy and electrochemistry can make individual nanoparticles in a liquid medium visible. The technique is suited to determine the activity of catalysts during their use. more

    Vibrational spectroscopy - Label-free imaging

    Spectroscopic methods are now granting us deep insights into biological systems at previously unattainable spatial and temporal resolutions. Complementing the already well-established fluorescence spectroscopy, the major potential of label-free vibrational spectroscopy has become clear in r ... more

  • Authors

    Kevin Wonner

    Kevin Wonner, born in 1995, studied chemistry with the focus on electrochemical nanoparticle characterization at the Ruhr University Bochum. He started his PhD in 2018 at the chair of Analytical Chemistry II of Professor Dr. Kristina Tschulik and is supported by the graduate school 2376. Hi ... more

    Mathies V. Evers

    Mathies Evers, born in 1989, studied chemistry at the Ruhr University Bochum, where he researched the synthesis of atom-precise molecular clusters. After his master's degree he started his doctoral thesis at the Chair of Analytical Chemistry II of Professor Dr. Kristina Tschulik and is supp ... more

    Prof. Dr. Kristina Tschulik

    Kristina Tschulik received her doctorate from TU Dresden in 2012 and worked as a postdoctoral fellow at the Leibniz Institute for Solid State and Materials Research Dresden and at the University of Oxford. Afterwards she established the working group for “Electrochemistry and Nanoscale Mate ... more

q&more – the networking platform for quality excellence in lab and process

The q&more concept is to increase the visibility of recent research and innovative solutions, and support the exchange of knowledge. In the broad spectrum of subjects covered, the focus is on achieving maximum quality in highly innovative sectors. As a modern knowledge platform, q&more offers market participants one-of-a-kind networking opportunities. Cutting-edge research is presented by authors of international repute. Attractively presented in a high-quality context, and published in German and English, the original articles introduce new concepts and highlight unconventional solution strategies.

> more about q&more

q&more is supported by:

 

Your browser is not current. Microsoft Internet Explorer 6.0 does not support some functions on Chemie.DE