Conformational studies of parathyroid hormone (PTH) PTH-related protein (PTHrP) chimeric peptides

The N-terminal 1-34 segments of both parathyroid hormone (PTH) and parathyr oid hormone-related protein (PTHrP) bind and activate the same membrane-emb edded G protein coupled receptor (PTH1 Rc) present on the surface of cells in target tissues such as bone and kidney. This binding occurs in spite of major differences between the two hormones in their amino acid sequence. Re cently, it was shown that in (1-34) PTH/PTHrP hybrid peptides, the N-termin al 1-14 segment of PTHrP is incompatible with the C-terminal 15-34 region o f PTH in terms of bioactivity. The sires of incompatibility were identified at positions 5 in PTHrP and 19 in PTH. In the present paper we describe th e synthesis biological evaluation, and conformational characterization of t wo segmental hybrids: PTHrP(1-27)-[Tyr(34)]bPTH(28-34)-NH2 (hybrid I) and P THrP(1-18)-[Nal(23), Try(34)]bPTH(19-34)-NH2 (hybrid II). Hybrid I is as ac tive as PTH(1-34)NH2 and more than two orders of magnitude more active than hybrid II. The conformational properties of the the hybrids were studied i n water/trifluoroethanol (TFE) mixtures and in aqueous solutions containing dodecylphosphocholine (DPC) micelles by CD, two-dimensional nmr and comput er simulations. Upon addition of TFE to the aqueous solution, both hybrids undergo a coil-helix transition. The helix content in 1:1 water/TFE obtaine d by CD data is about 75% for both hybrids. In the presence of DPC, helix f ormation is observed at detergent concentrations above critical micellar co ncentration and the maximum helix content is of similar to 35 and similar t o 30% for hybrid I and II, respectively. Combined nmr analysis, distance ge ometry, and molecular dynamics calculations suggest that, in both solvent s ystems, the biologically active hybrid I exhibits two flexible sites, cente red at residues 12 and 19 connecting helical segments. The flexibility poin t at position 19 is not present in the poorly active hybrid II. Our finding s support the hypothesis, proposed in our previous work, that in bioactive PTH analogues the presence and location of flexibility points between helic al segments ni-e essential for enabling them to Sold into the bioactive con formation upon interaction with the PTH1 receptor: (C) 2000 John Wiley & So ns, Inc.