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Conformational studies of parathyroid hormone (PTH)/PTH‐related protein (PTHrp) chimeric peptides

Conformational studies of parathyroid hormone (PTH)/PTH‐related protein (PTHrp) chimeric peptides The N‐terminal 1–34 segments of both parathyroid hormone (PTH) and parathyroid hormone‐related protein (PTHrP) bind and activate the same membrane‐embedded 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. Recently, it was shown that in (1–34) PTH/PTHrP hybrid peptides, the N‐terminal 1–14 segment of PTHrP is incompatible with the C‐terminal 15–34 region of PTH in terms of bioactivity. The sites of incompatibility were identified at positions 5 in PTHrP and 19 in PTH. In the present paper we describe the synthesis, biological evaluation, and conformational characterization of two segmental hybrids: PTHrP(1–27)‐(Tyr34)bPTH(28–34)‐NH2 (hybrid I) and PTHrP(1–18)‐(Nal23, Tyr34)bPTH(19–34)‐NH2 (hybrid II). Hybrid I is as active as PTH(1–34)NH2 and more than two orders of magnitude more active than hybrid II. The conformational properties of the hybrids were studied in water/trifluoroethanol (TFE) mixtures and in aqueous solutions containing dodecylphosphocholine (DPC) micelles by CD, two‐dimensional nmr and computer simulations. Upon addition of TFE to the aqueous solution, both hybrids undergo a coil–helix transition. The helix content in 1:1 water/TFE obtained by CD data is about 75% for both hybrids. In the presence of DPC, helix formation is observed at detergent concentrations above critical micellar concentration and the maximum helix content is of ∼35 and ∼30% for hybrid I and II, respectively. Combined nmr analysis, distance geometry, and molecular dynamics calculations suggest that, in both solvent systems, the biologically active hybrid I exhibits two flexible sites, centered at residues 12 and 19, connecting helical segments. The flexibility point at position 19 is not present in the poorly active hybrid II. Our findings support the hypothesis, proposed in our previous work, that in bioactive PTH analogues the presence and location of flexibility points between helical segments are essential for enabling them to fold into the bioactive conformation upon interaction with the PTH1 receptor. © 2000 John Wiley & Sons, Inc. Biopoly 54: 429–447, 2000 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Biopolymers Wiley

Conformational studies of parathyroid hormone (PTH)/PTH‐related protein (PTHrp) chimeric peptides

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Publisher
Wiley
Copyright
Copyright © 2000 John Wiley & Sons, Inc.
ISSN
0006-3525
eISSN
1097-0282
DOI
10.1002/1097-0282(200011)54:6<429::AID-BIP70>3.3.CO;2-N
Publisher site
See Article on Publisher Site

Abstract

The N‐terminal 1–34 segments of both parathyroid hormone (PTH) and parathyroid hormone‐related protein (PTHrP) bind and activate the same membrane‐embedded 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. Recently, it was shown that in (1–34) PTH/PTHrP hybrid peptides, the N‐terminal 1–14 segment of PTHrP is incompatible with the C‐terminal 15–34 region of PTH in terms of bioactivity. The sites of incompatibility were identified at positions 5 in PTHrP and 19 in PTH. In the present paper we describe the synthesis, biological evaluation, and conformational characterization of two segmental hybrids: PTHrP(1–27)‐(Tyr34)bPTH(28–34)‐NH2 (hybrid I) and PTHrP(1–18)‐(Nal23, Tyr34)bPTH(19–34)‐NH2 (hybrid II). Hybrid I is as active as PTH(1–34)NH2 and more than two orders of magnitude more active than hybrid II. The conformational properties of the hybrids were studied in water/trifluoroethanol (TFE) mixtures and in aqueous solutions containing dodecylphosphocholine (DPC) micelles by CD, two‐dimensional nmr and computer simulations. Upon addition of TFE to the aqueous solution, both hybrids undergo a coil–helix transition. The helix content in 1:1 water/TFE obtained by CD data is about 75% for both hybrids. In the presence of DPC, helix formation is observed at detergent concentrations above critical micellar concentration and the maximum helix content is of ∼35 and ∼30% for hybrid I and II, respectively. Combined nmr analysis, distance geometry, and molecular dynamics calculations suggest that, in both solvent systems, the biologically active hybrid I exhibits two flexible sites, centered at residues 12 and 19, connecting helical segments. The flexibility point at position 19 is not present in the poorly active hybrid II. Our findings support the hypothesis, proposed in our previous work, that in bioactive PTH analogues the presence and location of flexibility points between helical segments are essential for enabling them to fold into the bioactive conformation upon interaction with the PTH1 receptor. © 2000 John Wiley & Sons, Inc. Biopoly 54: 429–447, 2000

Journal

Biopolymers – Wiley

Published: Nov 1, 2000

Keywords: parathyroid hormone; parathyroid hormone related protein; chimeric peptides; nuclear magnetic resonance; conformation; micelles

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