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Cell-cell communication via endogenous peptides and their receptors is vital for controlling all aspects of human physiology and most peptides signal through G protein-coupled receptors (GPCRs). Disordered peptides bind GPCRs through complex modes for which there are few representative crystal structures. The disordered peptide neurotensin (NT) is a neuromodulator of classical neurotransmitters such as dopamine and glutamate, through activation of neurotensin receptor 1 (NTS 1 ). While several experimental structures show how NT binds NTS 1 , details about the structural dynamics of NT during and after binding NTS 1 , or the role of peptide dynamics on receptor activation, remain obscure. Here saturation transfer difference (STD) NMR revealed that the binding mode of NT fragment NT10-13 is heterogeneous. Epitope maps of NT10-13 at NTS 1 suggested that tyrosine 11 (Y11) samples other conformations to those observed in crystal structures of NT-bound NTS 1 . Molecular dynamics (MD) simulations confirmed that when NT is bound to NTS 1 , residue Y11 can exist in two χ 1 rotameric states, gauche plus (g + ) or gauche minus (g - ). Since only the g + Y11 state is observed in all the structures solved to date, we asked if the g - state is important for receptor activation. NT analogues with Y11 replaced with 7-OH-Tic were synthesized to restrain the dynamics of the side chain. P(OH-TIC)IL bound NTS 1 with the same affinity as NT10-13 but did not activate NTS 1 , instead acted as an antagonist. This study highlights that flexibility of Y11 in NT may be required for NT activation of NTS 1 ., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)