NMR-BASED STRUCTURAL STUDIES OF THE PNR-2 PS2 SINGLE-DOMAIN TREFOIL PEPTIDE - SIMILARITIES TO PORCINE SPASMOLYTIC PEPTIDE AND EVIDENCE FOR A MONOMERIC STRUCTURE/

NMR spectroscopy measurements have been used to obtain structural info rmation about the pNR-2/pS2 single-domain trefoil peptide. NMR data fr om 2D (two dimensional) double-quantum-filtered correlation spectrosco py (DQF-COSY), total correlation spectroscopy (TOCSY), NOE spectroscop y (NOESY), rotating frame NOE spectroscopy (ROESY) and 2D C-13-H-1 het eronuclear single-quantum coherence (HSQC) and C-13-H-1 HSQC-TOCSY spe ctra have been analysed to provide essentially complete H-1 and C-13 s equence-specific assignments for the pNR-2/pS2 protein. From a conside ration of the NOE intensities, (3)J(NH-alpha CH) coupling constants, H -1 and C-13 chemical shifts of backbone atoms and amide-proton exchang e rates, the pNR-2/pS2 was found to contain two short antiparallel bet a-strands (32-35 and 43-46), a short helix (25-30) and a type I beta-t urn (11-15). These elements of secondary structure are very similar to those found in the two trefoil domains of pSP for which detailed stru ctural information is already available. Similar H-1 chemical shifts w ere noted for several conserved residues in pNR-2/pS2 and pSP and a ch aracteristic Phe residue with a slowly flipping ring was found in the pNR-2/pS2 variant and in both domains of pSP. The tertiary structures of the domains therefore appear to be very similar in the two proteins and it is likely that the pNR-2/pS2 has the same pattern of disulphid e bonds (1-5, 2-4, 3-6) as pSP. Correlation time measurements derived from H-1-H-1 NOE measurements indicate that the Cys58-->Ser form of th e pNR-2/pS2 protein used in this study is monomeric in solution at app roximately 2 mM.