Probing transmembrane domain connecting loops in 7TM receptors to understand function

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. We have been able to express some (a handful) of them in simple E. coli bacterial cells and in virus cells as 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.

The information that is missing or difficult to obtain, is a description of the flexible or disordered loops of the proteins which extend beyond the membrane and determine selectivity and functional signalling. To obtain this information, we will use methods that can measure distances at the nanoscale (0.5-8nm +/- 0.01nm) in these receptors, as well as the time scale (in microseconds - nanoseconds) of the flexibility. Since this protein normally sits in a membrane, it needs some of the lipid components of the membrane to function properly, and we will investigate the receptor in its natural environment where it is functional. All the information from this cutting edge project will add to our general understanding of how they work, and help in future drug design and disease control when extended to other proteins.

Technical Summary

The loops of membrane proteins constitute both their specificity and interface to the outside environment, as well as communicate a signal to the cell. Since they have inherent mobility and disorder, which is recognised as being functionally important, they are often less well recognised or defined structurally. Solution state NMR has become the standard approach to define conformational excursions of biomolecules, but membrane-embedded proteins have effective molecular masses of MDa, when embedded in proteoliposomes - the receptors themselves are ~30-40kDa, but it is the complex size and form that restricts structural studies by solution state NMR (slow tumbling).

Solid state NMR (ssNMR) is ideally placed to probe membrane protein loops for several reasons, not least because of its applicability to large systems (no molecular weight limit), versatility in system morphology (membrane, bicelles, nanocrystals, etc.), as long as the information can be filtered through labelling. There are now several examples where the conformation of small receptor bound peptides and loops of proteins in large complexes have been resolved, and the application of ssNMR to insoluble proteins, including amyloid fibres, bound receptors and activating ligands bound to receptors, is a new field of research, when the expertise and instrumentation are in place.

Here, we will build on some good preliminary data obtained for an NMR labelled 7TM receptor in membranes, in which sample form has been optimized to yield (for the first time) ssNMR suitable for assignments of loop resonances (started under a previous MRC grant). This data is ready for assignments and will form the basis of conformational analyses focusing on loop sequences (unique short sequences will be labelled and picked out from the background; water-edited correlation spectra in reverse labelled receptor), with a goal of selecting out loop conformations in defined (protein-protein contact sites, ligand-binding sites) sites of the receptor. This information can then supplement other high resolution structural data to aid models for function, recognition and signalling.

The final goal (beyond this grant) is to further our understanding of how extra- and intra-cellular loops define descriptions of type, recognition and signalling for type A ligand-activated GPCRs. New insights into the mechanisms, how specificity is determined and how the signals are communicated to other (G-proteins) components will then be revealed.


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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 SS NMR 
Description Solid state NMR can be used on biological samples of any MWt, and in almost any form from crystals to heterogeneous membranes, as we have shown. The new approach here is with 2D crystalline proteins to resolve resonance from loops between helices, for the first time. Loops are most likely of significant importance for signalling and downstream processing of signals, and are so very important but often not resolved in crystal structures in a dynamic way, as with ss NMR. 
Type Of Material Technology assay or reagent 
Provided To Others? Yes  
Impact Now, membrane proteins can be studied in membranes. 
Description NMR assignments for membrane proteins 
Organisation University of Illinois at Urbana-Champaign
Department The Department of Chemistry
Country United States 
Sector Academic/University 
PI Contribution We provided samples with hitherto unachieved spectral resolution for pulse method development
Impact Angew Chem Int Ed Engl. 2011 Jul 18. doi: 10.1002/anie.201100730. [Epub ahead of print] The Conformation of Bacteriorhodopsin Loops in Purple Membranes Resolved by Solid-State MAS NMR Spectroscopy. Higman VA, Varga K, Aslimovska L, Judge PJ, Sperling LJ, Rienstra CM, Watts A.
Start Year 2009