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Biophysical Studies of the G-Protein Coupled Neurotensin Receptor

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Biophysical Studies of the G-Protein Coupled Neurotensin Receptor Biophysical Studies of the G-protein coupled Neurotensin Receptor Timothy Charles Hadingham Thesis submitted to the Board of the Faculty of Biological Sciences In partial fulfilment of the requirements for the degree of Doctor of Philosophy Pembroke College, Oxford 2006 Abstract Neurotensin (NT) is an endogenous tridecapeptide neurotransmitter (pGlu-Leu-Tyr-Glu-Asn- Lys-Pro-Arg-Arg-Pro-Try-Ile-Leu-OH) (1673 Da), whose effects when administered centrally include analgesia, inhibition of food intake, modulation of dopaminergic systems, modulation of pituitary hormone secretion, regulation of digestive function, and it acts as a trophic factor on human colon, pancreatic, prostate, and lung cancer cell lines. Its receptor protein, the G- protein coupled neurotesin 1 (NTS1), therefore represents an interesting target for the treatment of pain, obesity, schizophrenia, and cancer. Here, NTS1 has been expressed in .coli in the form of an N-terminally truncated fusion protein (NTS1-A) with maltose binding protein (N-terminally) and thyrodoxin (C-terminally) with typical yields of 2.7 nmols ± 2.3 nmols of active NTS1-A per 20 g of wet cell base and a novel protocol for the reconstitution of the receptor into a model membrane was elucidated. Solid phase peptide synthesis was used to produce NT and a variety of derivative peptides of NT to facilitate the studies of the receptor carried out in this thesis. Surface plasmon resonance (SPR) was utilised to study the kinetics of the interaction of NTS1-A with its ligand NT, using the receptor molecule as the analyte in order to enhance the response on binding; the data yielded an overall apparent KD in the nanomolar range of 37.3 ± 11.3 nmols. SPR was also applied in a new way to investigate techniques for the direct kinetic study of reconstituted receptor. Electron spin resonance (ESR) was used to study the dynamics of NT bound to NTS1-A, where it was demonstrated that there is an induced restriction of motion in the N-terminal region of NT upon binding to NTS1-A, with the correlation time increasing approximately by an order of magnitude at least (1.9 ns to 11.4 ns) in the presence of excess receptor binding sites. This study has suggested that an area of the peptide previously believed to have little biological relevance is, in fact, implicated in binding. Solid state nuclear magnetic resonance (NMR) was used to investigate 5 the assignment of resonances arising from labelled NT bound to NTS1-A. Using the R14 4 single quantum-double quantum correlation (1Q-2Q) experiment it has been possible to filter resonances arising from labelled ligand from the natural abundance background. This is the first time that this type of experiment has been used to study a ligand bound to a membrane protein. Finally, in order to investigate a possible solution to the difficulties in obtaining crystals of membrane proteins, NTS1-A was used as a test subject with repeating arrays of DNA Holliday junctions, with covalently attached NT. The success of this method was shown in the production of 2D crystals of NTS1-A, and were validated by electron microscopy. Contents Chapter 1 Introduction...........................................................................16 1.1 Biological Membranes................................................................................................16 1.1.1 The Pivotal Role of Membrane Proteins...................................................17 1.2 G-Protein-Coupled Receptors..................................................................................18 1.2.1 Neurotensin receptors (NTS).....................................................................21 1.2.2 Neurotensin ...................................................................................................22 1.2.3 Nonpeptide NT Antagonists.......................................................................24 1.2.4 NT structure-activity relationships.............................................................25 1.2.5 Mutagenesis and modelling of NTS1.........................................................27 1.3 Aims of the Thesis......................................................................................................33 Chapter 2 M aterials and M ethods..........................................................34 2.1 Aims of this Chapter..................................................................................................34 2.2 Materials.......................................................................................................................34 2.2.1 Bacterial growth media.................................................................................34 2.2.2 Antibiotic stock solution..............................................................................35 2.2.3 Reagents and buffers for the purification of NTS1-A ............................35 2.2.4 Buffers for SDS-PAGE gels and W estern blotting.................................36 2.3 Methods .......................................................................................................................36 2.3.1 Molecular Biology.........................................................................................36 2.3.2 Expression and Purification methods for NTS1-A.................................37 2.3.3 SDS-PAGE Gels...........................................................................................38 2.3.4 Western Blotting...........................................................................................39 2.3.5 Protein Assay.................................................................................................40 2.3.6 Binding assay of NT binding to NTS1-A..................................................41 - iii - 2.4 Reconstitution of NTS1-A into model membranes..............................................42 2.4.1 Preparation of brain polar lipids (BPL) liposomes ..................................42 2.4.2 BPLs liposome detergent titrations............................................................43 2.4.3 Reconstitution of NTS1-A into destabilised lipid vesicles meditated by Bio Beads .......................................................................................................43 2.5 Peptide synthesis.........................................................................................................44 2.5.1 Introduction...................................................................................................44 2.5.2 Methodology..................................................................................................46 2.6 Surface Plasmon Resonance (SPR)..........................................................................49 2.6.1 Immobilisation of ligand to sensor surfaces .............................................49 2.7 Electron Spin Resonance ..........................................................................................49 2.7.1 Spin labelling cystinylated peptides ............................................................49 2.7.2 Preparation of samples for ESR.................................................................50 2.7.3 ESR measurements.......................................................................................50 2.8 NMR.............................................................................................................................50 2.8.1 One pulse with decoupling..........................................................................50 2.8.2 Cross Polarisation Magic Angle Spinning (CP MAS)..............................51 5 13 2.8.3 R14 4 two dimensional C Carbon-Carbon Correlation Experiment...52 2.9 DNA arrays .................................................................................................................54 2.9.1 Preparation of DNA arrays .........................................................................54 2.9.2 Spectrophotometric analysis of array annealing.......................................54 2.9.3 Electron microscopy and image processing..............................................54 Chapter 3 Expression, Purification and Reconstitution of the G-Protein Coupled Receptor (GPCR) NTS1..........................................56 3.1 Introduction ................................................................................................................56 3.1.1 Expression of GPCRs..................................................................................56 3.1.2 Expression of NTS1.....................................................................................57 3.1.3 Aims of this chapter.....................................................................................59 - iv - 3.2 Synthesis of Biotinylated NT (Bio-NT)..................................................................59 3.3 Expression of NTS1-A in . coli DH5B in shaker flasks......................................61 3.3.1 Transformation .............................................................................................61 3.3.2 Growth...........................................................................................................61 3.3.3 Purification of NTS1-A Fusion Protein from flask scale growths ........61 3.3.4 Results from expression of NTS1-A in E.coli DH5B in shaker flasks...62 3.4 Large-scale fermenter production of NTS1-A in E. coli DH5B..........................66 3.4.2 500l Fermentation of pNTS1-A in DH5B................................................68 3.4.3 Discussion......................................................................................................75
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