Ma. Jimenez et al., HELIX FORMATION BY THE PHOSPHOLIPASE A(2) 38-59-FRAGMENT - INFLUENCE OF CHAIN SHORTENING AND DIMERIZATION MONITORED BY NMR CHEMICAL-SHIFTS, Biopolymers, 34(5), 1994, pp. 647-661
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.