Large protein complex observed at atomic level in the bacterial cell wall

A team of researchers at Utrecht University succeeded in zooming in on the atomic structure of a very large protein complex, which is used by bacteria to secrete substances into other bacterial or human cells. Using a novel approach, the researchers could observe parts of the complex in its natural environment that were previously inaccessible to other techniques. Marc Baldus, head of the NMR team and Professor of Structural Biology at Utrecht University, states: "Our ultimate goal is to observe these kind of protein complexes at work in the cell. With this study we made a big step in that direction". The results of this study is published in the journal Nature Methods.

All processes inside the cells of humans, animals, plants and microorganisms are carried out by a multitude of proteins. The contraction of a single muscle, for example, entails the concerted actions of thirty proteins. Characterizing these proteins and unravelling the way they are working together is crucial for understanding both health and disease. A critical step towards this goal involves obtaining and interpreting intricate atomic-level structural information on the proteins and their complexes.

NMR

The Utrecht research team employed nuclear magnetic resonance (NMR) for their studies. Similar to MRI, NMR uses the magnetic properties of nuclei to obtain structural information at atomic level. In the current study traditional NMR was combined with another technique that dramatically increases the sensitivity of the measurements by exploiting the much stronger magnetic properties of electrons.

Sensitive electrons

"Nuclei are relatively insensitive to the magnetic field", explains Prof. Baldus. "Therefore you need to measure rather long to be able to observe such a large protein complex, which is only present at low concentrations in its native environment. Electrons, which we add to our samples attached to specific molecules, are much more sensitive to the magnetic field. As a result, you need less time to measure."

Many signals

A significant challenge to be able to study such a large protein complex in its native environment is the large amount of NMR signals that will be observed. These signals not only stem from the many atoms of the protein complex itself, but also from molecules that form the native environment, such as lipids and other proteins. Thanks to the tailored methods developed by the Utrecht researchers, it is possible to zoom in on specific regions of the protein complex by making only them magnetically active.

NMR in Utrecht

In order to make use of the higher sensitivity of the electrons one needs a very powerful microwave source - a gyrotron - that is able to excite them. Thanks to funding obtained via the NWO VICI and NWO Groot programmes, the NMR department at Utrecht University owns an NMR spectrometer, with a 800MHz magnet, coupled to a gyrotron that is 200 times more powerful than regular microwave ovens. This is the world’s strongests setup of this kind. Hence these NMR studies are now possible providing unique opportunities to observe this type of protein complexes at work directly inside the cell.

Publication

Probing a cell-embedded Megadalton protein complex by DNP-supported solid-state NMR
Mohammed Kaplan*, Abhishek Cukkemane*, Gydo C.P. van Zundert*, Siddarth Narasimhan*, Mark Daniels*, Deni Mance*, Gabriel Waksman, Alexandre M.J.J. Bonvin*, Rémi Fronzes, Gert E. Folkers* and Marc Baldus*
Nature Methods, 18 May 2015, doi 10.1038/nmeth.3406
*Connected to Utrecht University

This study is closely related to Utrecht University’s strategic research theme Life Sciences.

More information

Monica van der Garde, press officer Utrecht University, Science Faculty, m.vandergarde@uu.nl, +31 (0)6 13 66 14 38.

Figure

NMR zooms with atomic detail in on a large, cell-embedded protein complex that bacteria use to secrete molecules. Orange and red balls show structural elements identified by NMR in a model that also includes microscopy and X-ray diffraction data previously obtained outside the cell environment.