The isolated C-terminal (F2) fragment of the Escherichia coli tryptophan synthase beta 2-subunit folds into a stable, organized nonnative conformation.
Proteolysis of the beta 2-subunit of Escherichia coli tryptophan synthase by the endoproteinase Glu C from Staphylococcus aureus V8 yields a peptide, F2, corresponding to the C-terminal 101 residues of the beta-chain. The conformation and stability of isolated F2 in phosphate buffer at pH 7.8 (where native beta 2 is stable) have been investigated. Circular dichroism spectra in the far-UV showed the presence of large amounts of secondary structure (19% alpha-helices, 34% extended beta-structures). Circular dichroism spectra in the near-UV and sedimentation velocity studies indicated an open globular structure with the aromatic side chains in a symmetric (or disordered) environment. NMR spectra and rates of amide proton exchange showed that F2 fluctuates rapidly between several conformations. The thermal denaturation of F2 observed by the loss of far-UV circular dichroism with increasing temperature appeared noncooperative, and indicates a high thermal stability (Tm = 70 degrees C). Differential scanning microcalorimetry confirmed the absence of cooperativity and indicated a very low value for the calorimetric enthalpy of denaturation (delta H = 17 kJ/mol). All these properties were compatible with a molten globule. However, the low sedimentation coefficient of F2 suggested a very hydrated and/or expanded structure, and the secondary structure content of isolated F2 (see above) differed widely from that reported in the literature for F2 within the context of native beta 2 (49% alpha-helices and 13% extended beta-structures). Thus, neither the secondary nor the tertiary structure of isolated F2 resembled those of native F2. In this respect, isolated F2 is not a "molten globule".(ABSTRACT TRUNCATED AT 250 WORDS)
Chaffotte
A
A
Unité de Biochimie Cellulaire, Institut Pasteur, Paris, France.
Guillou
Y
Y
Delepierre
M
M
Hinz
H J
HJ
Goldberg
M E
ME
D002942:Circular Dichroism; D010450:Endopeptidases; D004926:Escherichia coli / Q000201:enzymology*; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011487:Protein Conformation; D013816:Thermodynamics; D014367:Tryptophan Synthase / Q000737:chemistry*
D046911:Macromolecular Substances; D010446:Peptide Fragments; D010450:Endopeptidases; D014367:Tryptophan Synthase