Probing drug receptor binding sites driven by solid state NMR - An interdisciplinary approach.
Lead Research Organisation:
University of Oxford
Department Name: Biochemistry
Abstract
Biology works through highly synchronised chemical interactions at the atomistic scale. Small changes in the electronic charge of a biological molecule, or even the position of a hydrogen atom can have far-reaching consequences (e.g. the very contrasting actions of two subtly different forms (alpha, beta) of thalidomide, or a change of one amino acid in haemoglobin causing sickle cell anaemia). Such subtleties are becoming better understood at the molecular level, but still much is to be discovered and understood for promotion of health and well-being. The major class of targets in disease control for the next ten years is membrane proteins. These are a cell's first point of contact with the outside world and about 85% of all signals to the cell are transmitted through the membrane. It is not surprising then that both academics and drug companies are interested in how such signals are transmitted into the cell (2 Nobel prizes were awarded in 2005 for the revelation that one target membrane protein can activate and signal a multitude of other proteins, depending upon the nature of the small molecule activation, and over 10 Nobel prizes have been awarded for membrane protein studies since 1987). Membrane proteins are very difficult to work with, which is why there are only 21 structures (out of millions available) in the data bases. In addition, we do not have the structure of any ligand-activated human receptor. What we now need is a detailed insight into how these signals are initiated and transmitted at the molecular level, and this can be addressed using nuclear magnetic resonance (NMR) methods designed specifically for probing the detail at very high resolution (better than 0.03 nanometres) and with electronic and dynamic details but, very importantly, in the absence of the total structure of the target receptor protein.Solid state NMR exploits specifically the magnetic properties of some specific atoms for large heterogeneous, non-ordered macromolecules - this has been a fast growing area in structural biology and the UK is at the forefront of the developments. An essential part of this work is the incorporation of magnetic spies (or labels) into the molecule of interest so that we can obtain the information required. The chemical insertion of monitoring nuclei into the information-rich position in the macromolecule is vital and a pre-requisite and can only come from state-of-the-art clever chemistry directed at answering biologically important questions using physical methods. The NMR method is unique in producing very localized and highly specific information at a information-rich site, but this is only possible through the use of highly specialised chemistry to make molecules with the NMR labels where needed - hence this funding application will combine these two areas of expertise (NMR at Oxford and labelling at Bristol) to answer the important biological question How do small molecules activate proteins to transmit signals into a cell? . Detailed information gained will facilitate the understanding of, e.g. how a hormone causes a particular response, or how a toxic chemical initiates cell death. Importantly for wealth creation for the UK, which traditionally has been highly successful in discovering drugs, new design principles will be elucidated.
Organisations
People |
ORCID iD |
Anthony Watts (Principal Investigator) |
Publications
Iida K
(2002)
Synthesis of 5-[4,5- 13 C 2 ]- and 5-[1,5- 13 C 2 ]aminolevulinic acid
in Journal of Labelled Compounds and Radiopharmaceuticals
Varga K
(2007)
NMR crystallography: The effect of deuteration on high resolution 13C solid state NMR spectra of a 7-TM protein
in Biochimica et Biophysica Acta (BBA) - Biomembranes
Varga K
(2008)
Advances towards resonance assignments for uniformly--13C, 15N enriched bacteriorhodopsin at 18.8 T in purple membranes.
in Journal of biomolecular NMR
Attrill H
(2009)
Improved yield of a ligand-binding GPCR expressed in E. coli for structural studies.
in Protein expression and purification
Harding PJ
(2009)
Constitutive dimerization of the G-protein coupled receptor, neurotensin receptor 1, reconstituted into phospholipid bilayers.
in Biophysical journal
Tapaneeyakorn S
(2010)
Heterologous high yield expression and purification of neurotensin and its functional fragment in Escherichia coli.
in Protein expression and purification
Lam YH
(2010)
Solid-state NMR and simulation studies of equinatoxin II N-terminus interaction with lipid bilayers.
in Proteins
Grage SL
(2011)
Bilayer-mediated clustering and functional interaction of MscL channels.
in Biophysical journal
Judge PJ
(2011)
Recent contributions from solid-state NMR to the understanding of membrane protein structure and function.
in Current opinion in chemical biology
Pike KJ
(2012)
A spectrometer designed for 6.7 and 14.1 T DNP-enhanced solid-state MAS NMR using quasi-optical microwave transmission.
in Journal of magnetic resonance (San Diego, Calif. : 1997)
Cross TA
(2013)
Helical membrane protein conformations and their environment.
in European biophysics journal : EBJ
Description | This programme brought together synthetic chemistry and structural biology to gain a deeper understanding of important biological processes, the main focus being ligand activation of membrane receptors. This required the design and synthesis of small molecules, often incorporating reporter groups such as stable isotopes at specific sites, which can be visualised using NMR spectroscopy and, with very high magnetic fields, the local environment and dynamic behaviour can be detected. Using this strategy new insights are being gained into how proteins function. Amino acids are the building blocks of proteins and many stable labelled amino acids have been synthesised and used to investigate protein chemistry. For example, we are now able to probe the previously inaccessible regions of a light-detecting protein in bacteria. The protein is important as a model for the visual protein in the eyes of higher life forms. Understanding the subtleties of light reception is a major challenge and not described in detail so far. The methods we use probe directly the electronic configuration around the vitamin A derived light-activated molecule, retinal, and this is coupled to a specific and known location of an amino acid within the protein (at Lys216 in transmembrane helix 7). NMR allows precise probing of the lysine residue and the electronic changes that are crucial to activation to be described for the first time. For this project an efficient synthesis of [15N]-lysine was developed which delivered multi-gram quantities at ~10% of the cost of existing methods. The light receptor described above is similar in architecture to around 2000 brain receptors that are major targets in human therapy. Here we have used a similar strategy, but this time designed a selectively labelled activating small molecule of the receptor. We only know the structure and conformation of a handful of such receptors, and even less detail of the activating hormones and this is a novel approach to obtain the detail which is essential for future drug development. [1,3-13C2]-proline and [2-13C]-arginine were synthesised and incorporated into the ligand so we can describe the conformation, bent or straight, of this hormone for the first time. Extending the labelling methods to an enhancer of magnetic signals has also been achieved via synthesis of ligands with an inherent free electron. This special case gives rise to a magnetic signal which then passes its signal to the much less visible (nuclear) probes discussed above. This is very new technology and we only completed one data collection before the (home-built) equipment stopped working stably. Whilst a disappointment, the one set of data exceeds anything else reported worldwide with this sensitivity enhancement and technology. Sufficient signal enhancing compounds were prepared to allow many more experiments to be performed in the future once the instrumentation issues have been overcome. In addition to activation of membrane receptors described above, several smaller collaborative projects have been initiated leading to a greater understanding of metabolic processes. For example, fatty acid synthases (FASs) catalyse the biosynthesis of fatty acids, essential for all organisms. Structural analysis of FASs is crucial to understanding the mechanistic basis by which fatty acids are made. Acyl carrier protein (ACP) is the key player in this process. A series of substrates were synthesised and high resolution structures of these intermediates covalently bound to an ACP have been solved by solution NMR. These studies revealed that the ACP adopts a unique conformation for each intermediate driven by changes in the fatty acid binding pocket ensuring effective molecular recognition over subsequent rounds of fatty acid biosynthesis. In summary, the principle that designing the ideal molecule for use in exploring biological function, rather than using a less informative commercially available compound, has been proven. |
Exploitation Route | Drug companies making antimicrobial agents. Through design of novel antimicrobials. |
Sectors | Pharmaceuticals and Medical Biotechnology |
URL | http://www.bioch.ox.ac.uk/aspsite/index.asp?pageid=603 |
Description | We have made a number of isotopically labelled ligands for membrane drug targets and used them in investigations of their action. Some as still to be studied. |
First Year Of Impact | 2007 |
Sector | Chemicals,Healthcare |
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. |