Resolving structural details of membrane peptides and proteins at high resolution
Co-workers: Juan Bada Juarez, Juan Bolivar Gonzalez, Claudia Cassidy, Patricia Dijkman, Peter Fisher, Rosana Inácio dos Reis, Peter Judge, Steven Lavington, Juan Munoz-Garcia, Marc-Philipp Pfeil, Javier Vinals Camallonga, Daniel Yin.
How cells react to the external environment is still a major challenge in biology. Receptor and transporter proteins play pivotal roles in signalling and trafficking into and out of the cell. Smaller proteins and peptides can illicit dramatic changes in cellular behaviours, whether they are a signalling hormone, a component of an ion channel or induce structural changes such as cell death through lysis. We use a battery of biophysical methods to understand these processes in a quantitative way with a view to describing functional details that can ultimate lead to design of new therapies and infection control.
The neurotensin receptor (NTS1 a class of GPCR), play a pivotal role in neurotransmission, particularly disease and is a marker for colon cancer. We have expressed the protein in E. coli in structural biology amounts in a functionally competent form for structural studies, some of which involve single molecule approaches for bionanotechnological and drug design applications.
NTS1 is now available highly purified monodispersed in detergent and in a ligand-binding form. One approach to monitoring ligand binding has been to develop a novel surface plasmon resonance (SPR)method for tagging the natural ligand, neurotensin (13-mer peptide), to the chip and monitoring protein binding. Fluorescently tagged NTS1 has also been used in fluorescence resonance energy transfer methods to resolve long range information of protein-protein, induced signalling.
Using quantitative approaches such as single molecule studies a SPR and novel MST (microscale thermophoresis), we have been able to make an initial suggestion for the “GPCRInteractome”, to which further information can be added as it is obtained for this signalling system that is central to all eukaryote signalling processes and prime target for drug discovery.
Kinetics of the GPCR interactome. Showing some of the initial genetic steps in the GPCR signalling pathway.
Dijkman P.M., Watts A (2015) Lipid modulation of early G protein-coupled receptor signalling events, Biochimica et Biophysica Acta (BBA) - Biomembranes, Volume 1848, Issue 11, Part A, Pages 2889–2897
Adamson, R. and Watts, A. (2014) Kinetics of the early events of GPCR signalling, FEBS Letts,Volume 588, Issue 24, Pages 4701–4707
In the animal kingdom, especially reptile and fish small peptides act as antimicrobial agents, protecting against infection in the outer skin. Exploiting these fascinating properties, is something we have been focussing on recently. In particular, a new way of punching a hole into bacterial membranes, has been discovered. In delivering such peptides, we are exploiting polymer-stabilized lipid nano discs, about 10 nm (1,000 millionth of a metre) in diameter – there LipodisqTM particles also have potential for drug delivery and provide a membrane environment for membrane proteins without the use of detergents.
Publications - 2016
Bolivar JH, Munoz-Garcia JC, Castro-Dopico T, Dijkman PM, Stansfeld PJ, Watts A (2016), Interaction of lipids with the neurotensin receptor 1, BBA - Biomembranes, (in press)
- Kemp T.F., Dannatt H.R.W., Barrow N.S., Watts A., Brown S.P., Newton M.E., Dupree R. (2015), Dynamic Nuclear Polarization enhanced NMR at 187 GHz/284 MHz using an Extended Interaction Klystron Amplifier, J. Magnetic Resonance (in press)
Publications - 2015
- Dijkman P.M., Watts A (2015) Lipid modulation of early G protein-coupled receptor signalling events, Biochimica et Biophysica Acta (BBA) - Biomembranes, Volume 1848, Issue 11, Part A, Pages 2889–2897
- Dannatt H. R. W., Taylor G. F., Varga K., Higman V.A., Pfeil MP, Asilmovska L., Judge P. J., Watts A. (2015) C- and 1H-detection under fast MAS for the study of poorly available proteins: application to sub-milligram quantities of a 7 trans-membrane protein, Journal of Biomolecular NMR, Volume 62, Issue 1, pp 17-23
- Caoa Z., Dinga X., Penga B., Zhaob Y., Dingb J., Watts A., Zhao X. (2015) Novel expression and characterization of a light driven proton pump archaerhodopsin 4 in a Halobacterium Salinarum strain, BBA Bioenergetics, Volume 1847, Issues 4–5, Pages 390–398
- Fowler P.W., Orwick-Rydmark M., Radestock S., Solcan N., Dijkman P.M., Lyons J.A., Kwok J., Caffrey M., Watts, A. Forrest L.R., and Newstead S. (2015) Gating topology of the proton coupled oligopeptide symporters, Structure, Feb 3; 23(2): 290–301 (front cover feature).
- Goddard, A., Dijkman, P.M., Adamson, R.J., dos Reis, R.I. and Watts, A. (2014) Reconstitution of membrane proteins with a GPCR as an example, Methods in Enzymology, Volume 556, 2015, Pages 405-424
Publications - 2014
- Judge P. J., Taylor G.F., Dannatt H.R.W. and Watts A. (2015), “Solid-State Nuclear Magnetic Resonance Spectroscopy for Membrane Protein Structure Determination” CHAPTER 18 in Structural Proteomics: High-Throughput Methods, Methods in Molecular Biology, vol. 1261 (ed. R. J. Owens),
- Adamson, R. and Watts, A. (2014) Kinetics of the early events of GPCR signalling, FEBS Letts,Volume 588, Issue 24, Pages 4701–4707
- Liebel M, Schnedermann C, Bassolino G, Taylor G, Watts A, Kukura P (2014) Direct observation of the coherent nuclear response after the absorption of a photon. Physical Review Letters. ; 112, 238301
- Judge, P.J., Taylor, G. F.,Vermeer, L. S. and Watts A. (2014), "Structural Insights from Solid-State NMR into the Function of the Bacteriorhodopsin Photoreceptor Protein" CHAPTER 23 in Advances in Biological Solid-State NMR: Proteins and Membrane-Active Peptides, (eds F. Separovic, A. Naito)
Publications - 2013
- Goddard, A., Dijkman, P., Adamson, R., Watts, A. (2013) Lipid-Dependent GPCR Dimerization. In P. Michael Conn, editors: Receptor-Receptor Interactions, Vol 117, MCB, UK: Academic Press, pp. 341-357.
- Cross, T.A., Murray, D.T. and Watts, A. (2013) Helical Membrane Protein Conformations and their Environment, European Biophysics Journal, Vol. 42, Issue 10, pp 731-755
- Ryan, L., Lamarre, B., Diu, T., Ravi, J., Judge, P., Temple, A., Carr, M., Cerasoli, E., Su, B., Jenkinson, H., Martyna, G., Crain, J., Watts, A., Ryadnov, M. (2013) Anti-antimicrobial peptides: folding-mediated host defense antagonists. Journal of Biological Chemistry, 288, 20162-72.
- Long, A., O'Brien, C., Malhotra, K., Schwall, C., Albert, A., Watts, A., Alder, N. (2013) A Detergent-Free Strategy for the Reconstitution of Active Enzyme Complexes from Native Biological Membranes into Nanoscale Discs. BMC Biotechnology, 13, 41.
- Rakowska, P., Jiang, H., Ray, S., Pyne, A., Lamarre, B., Carr, M., Judge, P., Ravi, J., Gerling, U., Koksch, B., Martyna, G., Hoogenboom, B., Watts, A., Crain, J., Grovenor, C., Ryadnov, M. (2013) Nanoscale imaging reveals laterally expanding antimicrobial pores in lipid bilayers. Proc. Natl. Academ. Sci.(USA), 110, 8918-8923.
- Ding, X., Zhao, X., Watts, A. G-protein-coupled receptor structure, ligand binding and activation as studied by solid-state NMR spectroscopy. Biochem. J. 450, 443-457.
- Seidel, S., Dijkman, P., Lea, W., Bogaart, G., Jerabek-Willemsen, M., Lazic, A., Joseph, J., Srinivasan, P., Baaske, P., Simeonov, A., Katritch, I., Ladbury, J., Schreiber, G., Watts, A., Braun, D., and S. Duhr. (2013) Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions. Methods 59, 301-315.
Publications - 2012
- Orwick, M., Lovett, J., Graziadei, A., Lindholm, L., Hicks, M., Watts, A.(2012) Detergent-Free Incorporation of a Seven-Transmembrane Receptor Protein into Nanosized Bilayer Lipodisq Particles for Functional and Biophysical Studies. Nano Letters, 12, 4687-4692.
- Watts, A. (2012) Exploiting magnetic resonance spectral anisotropy averaging to gain biological details in biomembranes. Encyclopaedia of Magnetic Resonance – Historical Perspectives (E. D. Becker, Editor) Wiley Interscience.
- Higman, V. and Watts, A. (2012) "Recent Developments in Biomolecular Solid-State NMR", CHAPTER 13 in Recent Developments in Biomolecular NMR, (eds M. Clore and J. Potts).
- Oates, J., Faust, B., Attrill, H., Harding, P., Orwick, M., Watts, A. (2012) The role of cholesterol on the activity and stability of neurotensin receptor 1. BBA - Biomembranes, 1818, 2228-33.
- Goddard, A. and Watts, A. (2012) Contributions of fluorescence techniques to understanding G protein-coupled receptor dimerisation. Biophysical Review, 4, 291-298.
- Goddard, A. and Watts, A. (2012) Regulation of G protein-coupled receptors by palmitoylation and cholesterol. BMC Biology, 10, 27-30.
- Orwick, M., Judge, P., Procek, J., Lindholm, L., Graziadei, A., Engel, A., Grobner, G., Watts, A. (2012) Detergent-free formation and physico-chemical characterization of nanosized lipid-polymer complexes - Lipodisq. Angewante Chemie, 51, 1-6.
- Patil, A., Premaruban, T., Berthoumieu, O., Watts, A., Davis, J. (2012) Engineered Bacteriorhodopsin: A Molecular Scale Potential Switch. Chem. Eur. J., 18, 5632-36.
- Pike, K.J., Kemp, T.F., Takahashi, H., Day, R., Howes, A.P., Kryukov, E.V., MacDonald, J.F., Collis, A.E.C., Bolton, D.R., Wylde, R.J., Orwick, M., Kosuga, K., Clark, A.J., Idehara, T., Watts, A., Smith, G.M., Newton, M.E., Dupree, R., Smith, M.E. (2012) A spectrometer designed for 6.7 and 14.1 T DNP-enhanced solid-state MAS NMR using quasi-optical microwave transmission, J. Mag. Res., 215, 1-9.
- Berthoumieu, O., Patil, A., Wang, X., Aslimovska, L., Davis, J., and Watts, A. (2012) Molecular scale conductance photoswitching in engineered bacteriorhodopsin. Nano Letters, 12, 899–903.
- Patil, A., Premaruban, T., Berthoumieu, O., Watts, A., Davis, J. (2012) Enhanced photocurrent in engineered bacteriorhodopsin monolayer films.J. Phys. Chem. B, 116 , 683–689.
A Fascination with Vision - a video lecture for non-specialists
Research Images - drug and ligand binding
Figure 1: Substituted imidazo-pyridines are inhibitors of the gastric H+/K+-ATPase. By specific labelling of members of this drug family with NMR visible isotopes, we have been able to define the full conformation of the bound ligand, and suggest mechanism for inhibition from homology modelling with a related protein. (Kim, Watts & Watts, 2005, J. Med. Chem. 48, 7145-7152 and Watts, 2005, Nature Reviews Drug Discovery, 4, 555-568; Williamson et al., 2007, PNAS, 104, 18031-18036)
Figure 2: The cation-π interaction of acetyl choline, a major brain neurotransmitter, and the ligand gated, nicotinic acetyl choline receptor has been resolved using solid state NMR, giving an insight into the binding mechanism and the residues surrounding the site. (Watts, 2005, Nature Reviews Drug Discovery, 4, 555-568; Williamson et al., 2007, PNAS, 104, 18031-18036)
Figure 3: The way in which retinal is restrained within its binding site in membrane-embedded mammalian rhodopsin, and the structural details of the site, have been resolved for the early activation states of this light-activated GPCR, using high resolution solid state NMR to measure atomic distances within the retinal to high accuracy (+/- 0.2Å). (Spooner, et al., 2004, J. Mol. Biol., 343, 719-730)