Determination of the solution structure of neuropeptide K by high-resolution nuclear magnetic resonance spectroscopy.

1H NMR chemical shift assignments for neuropeptide K (NPK) and neurokinin A (NKA) have been determined at 600 MHz in 28% trifluoroethanol/water solution. Two-dimensional NMR techniques were used to assign proton resonances, and interproton distances were estimated from the observed nuclear Overhauser effects (NOEs). These distances were used as constraints in a simulated annealing protocol within the program XPLOR to generate structures consistent with experimental data. NPK forms a regular amphipathic alpha-helical structure from Asp 3, terminating at Gly 18. Slowly exchanging amide protons identified in this region are likely to be involved in hydrogen bonds to stabilize the helix. The remainder of the molecule displays many sequential NOEs, with some i-(i + 2) contacts, but little further evidence of defined secondary conformation. NKA displays strong sequential connectivities between amide protons from Thr 3 to Met 10, and some i-(i + 2) connectivities suggestive of a series of dynamic turns in equilibrium. A comparison of the tail region of NPK with the related peptide homologue, neurokinin A, in the same solvent system, indicates that both show increasing order when trifluoroethanol is titrated into water solution, with the appearance of sequential NOEs between backbone amide protons. Differences between the corresponding spans of primary sequence appear to be minimal. The clear finding that NPK adopts a well-defined helix in its N-terminal half and is relatively disordered in the C-terminal half, which includes the entire NKA sequence, may have important implications for understanding the increased selectivity of NPK over NKA for one class of neurokinin receptor.