An investigation into the conformational changes and lipid dependence of NTS1 activation by its agonist

Lead Research Organisation: University of Oxford
Department Name: Biochemistry


Many of our neurological functions are controlled through receptor proteins (called GPCRs) residing in the brain, and for this reason it has been estimated that about 30% of all drugs we use act on these receptors, of which there are more than 800. The functions controlled by GPCRs are numerous, and one receptor may be involved in many responses. We need to find out how these receptors work, and it has been said that this is the major challenge of current structural biology (Lagerstrom & Schioth, 2008, Nature Reviews Drug Discovery, 7, 339-57).

To help in that process of understanding, and as a result of recent breakthroughs, it is now possible to make a very limited number of GPCRs so that we can start to discover how they function. The one we have elected to work on is involved in pain management, neuromuscular diseases such as Parkinson s and the control of appetite, and hence obesity control. We have been able to express this mammalian brain receptor in simple E. coli bacterial cells, and it is functionally active to make any of the work we do relevant to its function in the brain. As with many of these receptors, they are activated by the binding of small molecules, and although we can monitor this binding, the important aspect is to understand and investigate how the protein is then activated and how it sends its signal to other proteins and then ultimately the cell.

We will, therefore, express and purify the receptor, activate it with hormone, and then use state-of-the-art methods to probe the receptor changes during the activation process. These methods can be used to measure distances at the nanoscale (0.5-8nm +/- 0.01nm) in these nanodetectors , as well as the time scale (in microseconds - nanoseconds) for the activation process. Since this protein normally sits in a membrane of the brain cell, it needs some of the lipid components of the membrane to function properly, and we will also investigate which of these components are required. Finally, if time permits, we will determine how the activated receptor passes the signal to the next component in the chain of signalling molecules, to unravel a little more about this vital signalling mechanism. All the information from this cutting edge project will add to our general understanding of brain signal transmission, and help in future drug design and disease control.

Technical Summary

The G-protein coupled receptor (GPCR), rat brain neurotensin receptor type I (NTS1) is one of a small number of GPCRs that have been successfully expressed in E.coli as a functional, ligand-binding receptor. The NTS1 receptor has been expressed and purified for lipid-protein reconstitutions by detergent removal at a range of molar ratios and characterized with density gradient centrifugation and ligand binding capacity.

Nitroxide spin-labels will be placed at either single sites or double cys sites on the receptor and ligand binding monitored. ESR spectra from labelled proteins are sensitive to backbone motions. Helical and beta-sheet sites having characteristic and more restricted motions than loop sites as a result of the differential anisotropy averaging of the nitroxide hyperfine interactions on a timescale (1/tau ~ (Azz - Axx,yy) ~ microsec - ns) which is relevant to backbone motions for isotropic proteins in solution such as a detergent solubilized receptor (r ~ 4nm). For membrane-embedded receptor, the spectra are additionally sensitive to probe motion due to being buried or accessible. Using this data, homology modeling of NTS1 will be based on the recently reported adrenergic receptors.

Doubly labeled NTS1 will then be studied to resolve the conformational changes in NTS1 on activation, by placing labels at the ends of helices to report on helical movements, as shown for rhodopsin on light activation. Distances of 1-8nm can be measured, and some indication of which helices are involved in activation of the receptor will be determined. About 8-10 such samples will be required, and a triangulation approach used to show how NTS1 is activated.

Finally, we will study the lipid dependence of NTS1 ligand binding and activation since we know that it is active in brain lipids, but not in any single component lipid studied. The possibility that cholesterol has a binding site will also be explored.
Description Structure-function studies of antimicrobial and fusogenic peptides by solid state NMR spectroscopy and MD simulation
Amount £434,931 (GBP)
Funding ID EP/I029516/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 02/2011 
End 01/2014
Title DEER 
Description ESR DEER is similar to FRET. The difference is that in FRET, two different labels are used but in DEER,m the labels are the same, but on different sites. Energy transfer between the labels os measured in pulsed ESR. The distance range is similar and not many pulsed ESR machines exist in the world for this kind of work, which is new. We now have much new data to be written up with this approach on membrane proteins. 
Type Of Material Technology assay or reagent 
Year Produced 2012 
Provided To Others? Yes  
Impact New data about how membrane proteins function is being provided with this methodolgy. 
Description Judge in Science Game and Course Advertising 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Worcester 6th Form College, judge in Science Game and course advertising

Elicited interest in science
Year(s) Of Engagement Activity 2012
Description Public lecture - on video and the WWW 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Primary Audience Public/other audiences
Results and Impact About 50 people atended, and the web lecture has been seen by others

A venture capital company approached me about commercialization of some of the work.
Year(s) Of Engagement Activity 2010