Polarization-Modulated FT-IR Spectroscopy Studies of Acetylcholinesterase Secondary Structure at the Air−Water Interface

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.

Surface FTIR Techniques to Analyze the Conformation of Proteins/ Peptides in H2O Environment

“…The technique of IRRAS for the Langmuir monolayer was developed in 1985 [33]. Subsequently, some proteins were shown to be also able to form a Lanmguir monolayer and IRRAS has been consequently used to analyze protein samples in the Langmuir monolayer [14,[34][35][36][37][38], which covers the surface of H 2 O. IRRAS can not only detect the FTIR signal of proteins, but also valuate the orientation of the proteins sample in the Langmuir monolayer due to the selection rules as discussed below [14,[34][35][36]38].…”

“…During the measurement of IRRAS of a Langmuir monolayer at the air-water interface, a vacuum cannot be applied and pure N 2 or dry air purging will not remove the water vapor from the continuous H 2 O evaporation of the water subphase. Although methods of delicate humidity control have been reported recently [38,39], a method able to automatically remove the water vapor IR absorption was welcomed and PM-IRRAS was developed as a consequence [34,45,46]. PM-IRRAS is similar to IRRAS and the difference between them stems from the polarization, which is switched between s-and p-polarization at a set frequency in PM-IRRAS.…”

“…Thus, any isotropic vibration (i.e., the vibration with randomly distributed orientation) will disappear. The vibrations of water vapor disappear in the PM-IRRAS results due to its isotropic orientation in the air [34,45,46]. When the incident angle is above the Brewster angle, the selection rule of PM-IRRAS is very similar to the p-polarized IRRAS: the peak will be positive if the vibration is parallel to the air-water interface.…”

“…By the above-mentioned surface FTIR methods, the amide I band of various conformations has been determined in an H 2 O environment [13,21,34,[53][54][55]. For example, the amide I band of α-helix is around 1650 cm -1 and that of unstructured conformation is around 1640 cm -1…”

“…The amide I band of β-sheet (at ~1630 cm -1 as well as ~1685 cm -1 ) and that of other conformations such as β-turns have been also determined [13,21,34,[53][54][55]. The position of the amide I band in H 2 O has been widely used as a reference to address the conformation of proteins in tissues and living cells [23,25,26], both of which are in H 2 O environment.…”