Proton nuclear magnetic resonance studies of intact native bovine parathyroid hormone.
Native intact bovine PTH was studied by proton nuclear magnetic resonance (NMR) techniques, at pH 3.5 and pH 6.3. The 1H-NMR spectra had good resolution and many multiplet structures were observed. Assignment of the NMR resonances corresponding to specific amino acids was approached using 1H chemical shifts, coupling constants, and pH dependence in the one-dimensional spectra and the 1H-1H connectivities revealed in two-dimensional homonuclear correlated spectroscopy (COSY) experiments. All the aromatic proton resonances were assigned. Two histidine residues had lower pK than the other two. The methyl groups of two residues were moved significantly downfield: using COSY and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) correlations, these were assigned to an alanine residue close to both Trp-23 and Tyr-43, and a valine residue in close spatial proximity to Trp-23. The NOESY spectrum also showed cross-peaks between the residues of the upfield valine-leucine-isoleucine methyl envelope. Many of the H alpha protons moved upfield as the pH was increased. These results indicate that intact native PTH exists in a preferred conformation in solution at pH 6.5. Our studies have provided new information on the three-dimensional spatial proximity of several amino acids along the polypeptide chain. The observed interactions are consistent with the currently accepted model suggesting that the hormone has two separate structural domains associated with the amino- and carboxy-terminal regions of the molecule respectively. The potential implications of this model for the expression of biological activity are discussed.
Coddington
J M
JM
Department of Chemistry, University of Auckland, New Zealand.
Barling
P M
PM
D000409:Alanine / Q000032:analysis; D000818:Animals; D002417:Cattle; D006639:Histidine / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D010281:Parathyroid Hormone / Q000032:analysis*; D011522:Protons
D010281:Parathyroid Hormone; D011522:Protons; D006639:Histidine; D000409:Alanine