Polarization-modulated FT-IR spectroscopy studies of acetylcholinesterase secondary structure at the air-water interface

Leila Dziri, Bernard Desbat, Roger M. Leblanc

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47 Scopus citations

Abstract

The vibrational spectrum of acetylcholinesterase (ACHE) at the air-water interface in its free form and bound to either its substrate, acetylthiocholine, or organophosphorus (OP) inhibitor has been studied by polarization modulation infrared reflection absorption spectroscopy (PMIRRAS). The shape and position of the amide I band was used to gauge the surface orientation of α-helices and β-sheets. The measured secondary structure content indicated that the enzyme did not unfold for the surface pressures used (0-30 mN/m). At low surface pressures, a strong amide I band indicated that the average tilt axis of the helices was aligned parallel to the air - water interface. Upon further compression, the α-helix component was significantly reduced, because the tilt axis of the helix relative to the water surface achieved a perpendicular orientation. PMIRRAS was also used to investigate the effect of phospholipids on molecular organization and orientation of AChE at the air - water interface. The enzyme was found to be fully inserted into the lipidic film during compression. The hydrolysis and inhibition were studied at the air - water interface. Band frequencies associated with acetylthiocholine binding to the enzyme active site and formation of the reaction products were observed. The OP inhibitor, paraoxon, was observed to unfold the enzyme at the air - water interface, because only high-frequency components associated with the extended conformation were observed upon compression. The secondary structure of the AChE was reestablished 30 min after a reactivator, trimethyl bis-(4-formylpyridinium bromide) dioxime, was injected beneath the paraoxon-inhibited ACHE. For the first time, an in situ study of the protein conformation is reported using the PMIRRAS technique, and direct supporting evidence that the enzyme did not lose its native secondary structure upon spreading at the air - water interface is provided.

Original languageEnglish (US)
Pages (from-to)9618-9625
Number of pages8
JournalJournal of the American Chemical Society
Volume121
Issue number41
DOIs
StatePublished - Oct 20 1999

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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