Structure of recombinant human parathyroid hormone in solution using multidimensional NMR spectroscopy.

Abstract

The solution structure of human parathyroid hormone, in the form of recombinant prolyl-hPTH(1-84), has been investigated by multidimensional NMR spectroscopy under conditions (aqueous trifluoroethanol) which favour the structured-state of the protein. Spin systems were identified from 3D 1H DQF (double-quantum filtered)-COSY and TOCSY spectra and sequence-specific assignments were from 2D 1H phase-sensitive NOESY spectra. Signal overlap was resolved in a 3D-NOESY-TOCSY spectrum and assignments were confirmed with 2D NOESY-15N-HMQC (heteronuclear multiple-quantum coherence) spectra taken of a sample universally labeled with 15N. A satisfactory set of final structures was calculated from the quantitative NOE data using restrained molecular dynamics and energy minimization calculations. The N-terminus is dominated by three, well defined helices between Ser-3 to Asn-10, Ser-17 to Lys-27 and Asp-30 to Leu-37, while the most significant structural features in the C-terminus are a short, less-well defined helix between Asn-57 to Ser-62 and a series of loose turns. These two terminal units are joined by an unstructured mid-region. The molecule shows a tendency towards tertiary structure, defined by a number of long-range NOEs. A detailed RMS deviation analysis allowed the final refined structures to be classified into a limited ensemble of stable conformations that reflect the inherent flexibility of the hormone in solution.