Probing the environment of neurotensin whilst bound to the neurotensin receptor by solid state NMR.
A functionally active analogue of neurotensin, neurotensin(8-13), has been observed whilst bound to the agonist-binding site of the rat neurotensin receptor by nuclear magnetic resonance (NMR). Through the application of slow magic angle sample spinning and high-power proton decoupling, sufficient resolution and sensitivity were obtained in the carbon-13 spectrum to allow an assignment of many of the side chain resonances arising from uniformly carbon-13/nitrogen-15-labelled neurotensin(8-13) whilst bound to the neurotensin receptor. Significant perturbations in carbon-13 chemical shift were observed upon the binding of the neurotensin(8-13) to the receptor. Most importantly significant shifts were observed in both the carboxy terminus and tyrosine side chain of the neurotensin(8-13), suggesting that these sites are important in the interaction of the neurotensin with the agonist-binding site on the neurotensin receptor. Conversely, no perturbations were observed for the carbon-13 sites within the guanidinium groups of the arginine side chains, indicating little interaction with the receptor-binding site, or a shielding of the local environment by the surrounding nitrogen atoms. These NMR observations lend further support to previous structure-activity studies, site-directed mutagenesis and modelling studies of the agonist-binding site of the neurotensin receptor, from which the same specific residues for which NMR perturbations were observed are important for neurotensin receptor activation by neurotensin.
Williamson
P T F
PT
Laboratorium für Physikalische Chemie, ETH-Hönggerberg, CH-8093 Zurich, Switzerland.
Bains
S
S
Chung
C
C
Cooke
R
R
Watts
A
A
D000818:Animals; D001665:Binding Sites; D009496:Neurotensin / Q000737:chemistry* / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D051381:Rats; D018028:Receptors, Neurotensin / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013329:Structure-Activity Relationship
D010446:Peptide Fragments; D018028:Receptors, Neurotensin; D009496:Neurotensin; C054381:neurotensin (8-13)