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 information about the pNR-2/pS2 single-domain trefoil peptide. NMR data from 2D (two dimensional) double-quantum-filtered correlation spectroscopy (DQF-COSY), total correlation spectroscopy (TOCSY), NOE spectroscopy (NOESY), rotating frame NOE spectroscopy (ROESY) and 2D 13C-1H heteronuclear single-quantum coherence (HSQC) and 13C-1H HSQC-TOCSY spectra have been analysed to provide essentially complete 1H and 13C sequence-specific assignments for the pNR-2/pS2 protein. From a consideration of the NOE intensities, 3J(NH-alpha CH) coupling constants, 1H and 13C chemical shifts of backbone atoms and amide-proton exchange rates, the pNR-2/pS2 was found to contain two short antiparallel beta-strands (32-35 and 43-46), a short helix (25-30) and a type I beta-turn (11-15). These elements of secondary structure are very similar to those found in the two trefoil domains of pSP for which detailed structural information is already available. Similar 1H chemical shifts were noted for several conserved residues in pNR-2/pS2 and pSP and a characteristic 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 disulphide bonds (1-5, 2-4, 3-6) as pSP. Correlation time measurements derived from 1H-1H NOE measurements indicate that the Cys58-->Ser form of the pNR-2/pS2 protein used in this study is monomeric in solution at approximately 2 mM.
Laboratory of Molecular Structure, National Institute for Medical Research, London, England.
V I
VI
Polshakov
T A
TA
Frenkiel
B
B
Westley
M
M
Chadwick
F
F
May
M D
MD
Carr
J
J
Feeney
D000595:Amino Acid Sequence; D000818:Animals; D006133:Growth Substances / Q000737:chemistry*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009077:Mucins; D009124:Muscle Proteins; D009363:Neoplasm Proteins / Q000737:chemistry*; D009479:Neuropeptides; D010455:Peptides / Q000737:chemistry*; D017433:Protein Structure, Secondary; D011506:Proteins; D016415:Sequence Alignment; D013552:Swine; D000071163:Trefoil Factor-1; D000071164:Trefoil Factor-2; D000071165:Trefoil Factor-3; D025521:Tumor Suppressor Proteins
D006133:Growth Substances; D036341:Intercellular Signaling Peptides and Proteins; D009077:Mucins; D009124:Muscle Proteins; D009363:Neoplasm Proteins; D009479:Neuropeptides; D010455:Peptides; D011506:Proteins; C051838:TFF1 protein, human; C099037:TFF3 protein, rat; D000071163:Trefoil Factor-1; D000071164:Trefoil Factor-2; D000071165:Trefoil Factor-3; D025521:Tumor Suppressor Proteins; C034407:pancreatic spasmolytic polypeptide