Neutron Scattering Provides New Insight into Protein Substrate Processing
Credit: Institut Laue-Langevin
AAA+ ATPases are a large family of ubiquitous enzymes with multiple tasks, including the remodelling of the cellular proteome. A subfamily, so-called unfoldases, recognize, unfold, and address misfolded or dysfunctional proteins towards proteolytic complexes, which eliminate these potentially toxic proteins for a healthy, functional state of the cellular proteome. Given the intrinsic flexibility of ATPases and the transient character of the interaction with their protein substrates, it is challenging in structural biology to follow the conformational changes of these enzyme-substrate complexes during the active unfolding process.
In a collaboration between ILL and IBS (Institut de Biologie Structurale) a novel approach was developed combining time-resolved small angle neutron scattering (TR-SANS) with online-fluorescence spectroscopy on D22, in order to monitor the PAN unfoldase from the deep-sea Methanocaldococcus jannaschii organism and a Green Fluorescent Protein (GFP) model substrate. By using alternating perdeuteration of both partners and by controlling the enzymatic activity of the hyperthermophilic PAN system by temperature activation at 55-60°C, it was possible to follow conformational changes of both PAN and GFP separately and individually during the active unfolding process at a time resolution of 30 seconds.
The results show the progressive unfolding and aggregation of GFP as well as a concomitant and reversible contraction of the PAN unfoldase during the active reaction. While the methodological approach developed has been designed for this specific project, it is expected that it can be applied to a wide range of biological macromolecular complexes, and provide structural information from individual partners at a time resolution of some seconds.
Ibrahim, Z., Martel, A., Moulin, M., Kim, H. S., Härtlein, M., Franzetti, B., & Gabel, F. (2017). Time-resolved neutron scattering provides new insight into protein substrate processing by a AAA+ unfoldase. Scientific Reports, 7, 40948. doi:10.1038/srep40948
This article has been republished from materials provided by the Institut Laue-Langevin . Note: material may have been edited for length and content. For further information, please contact the cited source.
How Food Poisoning Bacteria Campylobacter Uses Other Organisms as Trojan Horse to Infect New HostsNews
Campylobacter jejuni is one of the most common causes of gastroenteritis often infecting humans through raw or under-cooked poultry. The new study revealed how the bacteria can infiltrate micro-organisms called amoebae, multiplying within their cells while protected inside its host from harsh environmental conditions.READ MORE
Aquatic Plant May Help Remove Contaminants From LakesNews
Duckweed might help remove contaminants from ponds and slow-moving water-bodies and then could be harvested and incorporated into animal feed.READ MORE
Cleaning Okinawan Pig Farm Wastewater with Microbial Fuel CellsNews
Using a promising wastewater treatment technology, called a microbial fuel cell (MFC), new research could reduce the wastewater burden. Agricultural practices, such as pig farming, produce a large amount of wastewater containing organic contaminants, malodorous gases, and other substances that are damaging to the water supply.READ MORE