Protein stability and interaction of the nicotinic acetylcholine receptor with cholinergic ligands studied by Fourier-transform infrared spectroscopy

Abstract

Based on the conformational dependence of the amide-I i.r. band, this paper explores the use of Fourier-transform i.r. spectroscopy methods to probe structural features of proteins present in native membranes from Torpedo highly enriched in acetylcholine receptor (AcChR). The interference of water absorbance on the amide-I spectral region has been eliminated through isotopic exchange by freeze-drying the membranes in the presence of trehalose to avoid protein denaturation induced by drying, followed by resuspension in deuterated water. AcChR-rich membrane samples prepared in such a way maintained an ability to undergo affinity-state transitions and to promote cation translocation in response to cholinergic agonists, which are functional characteristics of native untreated samples. The temperature-dependence of the i.r. spectrum indicates a massive loss of ordered protein structure, occurring at temperatures similar to those reported for thermal denaturation of the AcChR by differential scanning calorimetry and by thermal inactivation of alpha-bungarotoxin-binding sites on the AcChR [Artigues, Villar, Ferragut & Gonzalez-Ros (1987) Arch. Biochem. Biophys. 258, 33-41], thus suggesting that the observed i.r. spectral changes correspond to alterations in the structure of the AcChR protein. Furthermore, the presence of detergents as well as cholinergic agonists and antagonists produces spectral changes that are also consistent with the alterations in AcChR protein structure expected from previous calorimetric studies. In contrast with the information obtained by calorimetry, i.r. spectroscopy allows the contribution of secondary structural changes to be distinguished from the overall change in protein structure. Thus prolonged exposure to cholinergic agonists, which drives the AcChR protein into the desensitized state, produces only negligible alterations in the amide-I band shape, but increases substantially the thermal stability of the protein. This suggests that rearrangements in the tertiary or quaternary structure of the protein are more likely to occur than extensive changes in secondary structure as a consequence of AcChR desensitization.

Thermal Stability and Aggregation of Sulfolobus solfataricus β-Glycosidase Are Dependent upon the N-∈-Methylation of Specific Lysyl Residues

“…Only small differences were noticed, by monitoring the amide IЈ bandwidth as a function of the temperature (32,33) at p 2 H 7.0, in order to determine the thermal denaturation curves of the two forms of the protein (data not shown). Since this method did not appear to be sufficiently sensitive to detect subtle changes in the secondary structure, a more detailed investigation of protein thermal denaturation was performed by difference spectroscopy (32,34,35).…”

“…A recent observation of high medical interest concerns the inhibition of toxicity of the synthetic ␤-amyloid peptide ␤- (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) by N-methylation (11).…”

Influence of ADP, AMP‐PNP and of depletion of nucleotides on the structural properties of F1ATPase: a Fourier transform infrared spectroscopic study

“…It should be noted that even in the AMP-PNP/FI spectrum the shoulder at about 1619 cm -t is absent as in the ADP/F 1 spectrum, indicating that protein intermolecular interactions are inhibited also by AMP-PNE Fig. 4 reports the thermal denaturation profiles of F~ATPase samples as monitored by the amide I' band-width as a function of temperature [20]. The figure shows that the temperature of maximum protein denaturation (TMPD), corresponding to the curve inflection points, occurs at about 50, 60 and 65°C in sol/F1, ADP/F~ and AMP-PNP/F~, respectively.…”

Mechanism of thermal denaturation of maltodextrin phosphorylase from Escherichia coli

“…Therefore, this finding indicates that the solvent (#H # O) has a smaller accessibility to MalP in the presence of 50 mM and 100 mM P i , compared with the soluble fraction of MalP in 2 mM P i . The bands shown in Figure 3A can be assigned according to previous studies on proteins [26][27][28][29][30][31]. In particular, the 1653n8 cm −" band is due to α-helical structure while β-sheets display a band at 1638 and 1639 cm −" .…”

“…The increase in temperature leads also to other changes in the infrared spectrum, such as, for instance, amide Ih band-shift and broadening [28]. The denaturation has been followed by monitoring as a function of the temperature, the amide Ih bandwidth, calculated at $ % of the amide Ih band height (W3\4H).…”