HELIX FORMATION BY THE PHOSPHOLIPASE A(2) 38-59-FRAGMENT - INFLUENCE OF CHAIN SHORTENING AND DIMERIZATION MONITORED BY NMR CHEMICAL-SHIFTS

The solution structure of a peptide fragment corresponding to the 38-5 9 region of porcine phospholipase A(2) has been investigated using CD, nmr chemical shifts, and nuclear overhauser effects (NOEs). This isol ated fragment of phospholipase forms an alpha-helix spanning residues 38-55, very similar to the one found in the native protein, except for residues 56-58, which were helical in the crystal but found random in solution. Addition of triflouro-ethanol (TFE) merely increased helix population but it did not redefine helix limits. To investigate how th e folding information, in particular that concerning eventual helix st art and stop signals, was coded in this particular amino acid sequence , the helices formed by synthetic peptides reproducing sections of thi s phospholipase 38-59 fragment, namely 40-59, 42-59, 38-50, and 45-57, were characterized using NOEs and helix populations quantitatively ev aluated on different peptide chain segments using nmr chemical shifts in two solvents (H2O and 30% TFE/H2O). A set of nmr spectra was also r ecorded and assigned under denaturing conditions (6M urea) to obtain r eliable values for the chemical shifts of each peptide in the random s tate. Based on chemical shift data, it was concluded that the helix fo rmed by the phospholipase 38-59 fragment was not abruptly, but progres sively, destabilized all along its length by successive elimination of residues at the N end, while the removal of residues at the C end aff ected helix stability more locally and to a lesser extent. These resul ts are consistent with the idea that there are not single residues res ponsible for helix initiation or helix stability, and they also eviden ce an asymmetry for contributions to helix stability by residues locat ed at the two chain ends. The restriction of molecular mobility caused by linking with a disulphide bridge at Cys 51 two identical 38-59 pep tide chains did not increase helix stability. The helix formed by the covalently formed homodimer was very similar in length and population to that formed by the monomer. (C) 1994 John Wiley and Sons, Inc.