Folding of the Squash Trypsin Inhibitor EETI II

Evidence of Native and Non-Native Local Structural Preferences in a Linear Analogue

Authors


Correspondence to A. Heitz, Centre de Biochimie Structurale, Faculté de Pharmacie, 15 Avenue Charles Flahault, F-34060 Montpellier Cedex 1, France

Abstract

A peptide, corresponding to the entire sequence of the squash trypsin inhibitor EETI II (Ecballium elaterium trypsin inhibitor) in which the six cysteines, engaged in three disulphide bridges in native EETI II, have been replaced by six serines, has been synthesised. CD, Fourier-transform infrared spectroscopy (FTIR) and 1H-NMR studies of this peptide revealed that some secondary structures present in native EETI II are still populated in the absence of disulphide bonds. Native-like secondary structures were observed for segments 10–15 (helix), 16–19 and 22–25 (reverse turns) but no native tertiary interaction was detected. However, a non-native local interaction between the aromatic ring of Phe26 and the amide group of Gly28 was observed. It is hypothesised that the 10–15, 16–19 and 22–25 native-like local conformations could play a major role in the early folding of EETI II.

Abbreviations
BPTI

bovine pancreatic trypsin inhibitor

CMTI

Cucurbita maxima trypsin inhibitor

COSY

correlation spectroscopy

DQF-COSY

double-quantum-filtered correlation spectroscopy

EETI

Ecballium elaterium trypsin inhibitor

FTIR

Fourier-transform infrared spectroscopy

NOESY

nuclear Overhauser effect spectroscopy

RMS

root mean square

ROESY

rotating-frame NOESY spectroscopy

TOCSY

total correlation spectroscopy

1D

one-dimensional

2D

two-dimensional

3D

three-dimensional

Ancillary