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Conformational studies of parathyroid hormone (PTH)/PTH‐related protein (PTHrP) point‐mutated hybrids
Author(s) -
Peggion Evaristo,
Mammi Stefano,
Schievano Elisabetta,
Behar Vered,
Rosenblatt Michael,
Chorev Michael
Publication year - 1999
Publication title -
biopolymers
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/(sici)1097-0282(19991015)50:5<525::aid-bip6>3.0.co;2-8
Subject(s) - chemistry , parathyroid hormone , parathyroid hormone related protein , peptide , micelle , valine , receptor , peptide sequence , amino acid , parathyroid hormone receptor , helix (gastropod) , arginine , aqueous solution , stereochemistry , biochemistry , hormone receptor , calcium , medicine , gene , organic chemistry , ecology , cancer , biology , breast cancer , snail
The N‐terminal 1–34 segments of both parathyroid hormone (PTH) and parathyroid hormone‐related protein (PTHrP) bind and activate the same membrane receptor in spite of major differences between the two hormones in their amino acid sequence. Recently, it was shown that in (1–34)PTH/PTHrP segmental hybrid peptides, the N‐terminal 1–14 segment of PTHrP is incompatible with the C‐terminal 15–34 region of PTH leading to substantial reduction in potency. The sites of incompatibility were identified as positions 5 in PTH and 19 in PTHrP. In the present paper we describe the synthesis, biological evaluation, and conformational characterization of two point‐mutated PTH/PTHrP 1–34 hybrids in which the arginine residues at positions 19 and 21 of the native sequence of PTHrP have been replaced by valine (hybrid V 21 ) and glutamic acid (hybrid E 19 ), respectively, taken from the PTH sequence. Hybrid V 21 exhibits both high receptor affinity and biological potency, while hybrid E 19 binds weakly and is poorly active. The conformational properties of the two hybrids were studied in aqueous solution containing dodecylphosphocholine (DPC) micelles and in water/2,2,2‐trifluoroethanol (TFE) mixtures. Upon addition of TFE or DPC micelles to the aqueous solution, both hybrids undergo a coil‐helix transition. The maximum helix content in 1 : 1 water/TFE, obtained by CD data for both hybrids, is ∼ 80%. In the presence of DPC micelles, the maximum helix content is ∼ 40%. The conformational properties of the two hybrids in the micellar system were further investigated by combined 2D‐nmr, distance geometry (DG), and molecular dynamics (MD) calculations. The common structural motif, consisting of two helical segments located at N‐ and C‐termini, was observed in both hybrids. However, the biologically potent hybrid V 21 exhibits two flexible sites, centered at residues 12 and 19 and connecting helical segments, while the flexibility sites in the weakly active hybrid E 19 are located at position 11 and in the sequence 20–26. Our findings support the hypothesis that the presence and location of flexibility points between helical segments are essential for enabling the active analogs to fold into the bioactive conformation upon interaction with the receptor. © 1999 John Wiley & Sons, Inc. Biopoly 50: 525–535, 1999