Fourier transform coupled to tryptophan-scanning mutagenesis: Lessons from its application to the prediction of secondary structure in the acetylcholine receptor lipid-exposed transmembrane domains

Abstract

Although Fourier transform (FT) and tryptophan-scanning mutagenesis (TrpScanM) have been extremely useful for predicting secondary structures of membrane proteins, they are deemed to be low-resolution techniques. Herein, we describe the combined use of FT and TrpScanM (FTTrpScanM) as a more reliable approach for the prediction of secondary structure. Five TrpScanM studies of the acetylcholine receptor lipid-exposed transmembrane domains (LETMDs) were revisited and analyzed by FT-TrpScanM. FT analysis of the raw data from the aforementioned TrpScanM studies supports and validates the conclusions derived from their tryptophanperiodicity profiles. Furthermore, by FT-TrpScanM, we were able to determine the minimum number of consecutive tryptophan substitutions necessary for more robust prediction of α-helical secondary structures and evaluate the quality of structure predictions by α-helical character curves. Finally, this study encourages future utilization of FT-TrpScanM to more reliably predict secondary structures of the membrane protein LETMDs.

A conserved cysteine residue in the third transmembrane domain is essential for homomeric 5-HT3receptor function

“…1B). While the resolution of this cryo-electron microscopy-derived model is relatively low, the periodicity of the α subunit's M3 helix agrees well with a nuclear magnetic resonance spectroscopy model of a synthetic peptide corresponding to the M3 domain (Lugovskoy et al 1998) and predictions made by Fourier transform analyses of functional data obtained with M3 Trp scanning mutagenesis (Otero-Cruz et al 2008). According to these predictions the M3 5 Thr residue in the nACh α subunit, corresponding to Cys-312 in the 5-HT 3 A subunit, faces towards the other membrane spanning domains within the same subunit.…”

Fourier transform coupled tryptophan scanning mutagenesis identifies a bending point on the lipid-exposed δM3 transmembrane domain of theTorpedo californicanicotinic acetylcholine receptor

“…Spatial restrictions at a labeling site The α-helical character curves are a novel predictor tool that has been used in the analysis of the secondary structure of various lipid-exposed TMDs. 36 The α-helical character curves do not display complete α-helical character, suggesting that the δM3 domain is structurally composed of a combination of different helix types: α-helix and 3 10 -helix ( Fig. 3C and F).…”

“…An important aspect of these peaks, identified by an asterisk in Figure 3B and E, is that, although they are not the predominant peaks due to the disparity in the oscillatory amplitudes of the tryptophan periodicity profiles, they represent the mean periodicities of the tryptophan periodicity profiles. 20,21,36 The mean periodicities estimated from the Fourier transform power spectra for the closed-and open-channel states were determined to be 2.96 and 2.63 amino acids per helical turn, respectively. These results correlate well with the mean periodicities obtained from the analysis of the tryptophan periodicity profiles ( Table 2).…”

“…This analysis was performed as described previously to validate the mean periodicities calculated using tryptophan-scanning mutagenesis. 21,34,36 α-helical character curves. α-Helical character curves were generated as described previously in reference 36.…”

Tryptophan scanning of the acetylcholine receptor's βM4 transmembrane domain: Decoding allosteric linkage at the lipid-protein interface with ion-channel gating

“…The periodicities of AChR expression (fmol/ Å 3 ) and ACh EC 50 (µM) periodicity profiles were also determined by Fourier transform power spectra. The Fourier transform power spectra from data of AChR expression and ACh EC 50 periodicity profiles were generated using a least-squares Fourier transform equation: 26,27,36,[53][54][55][56][57][58] (3)…”