Secondary structure and temperature behaviour of acetylcholinesterase

Abstract

The secondary structure of the acetylcholinesterase and its temperature behaviour have been investigated using Fourier-transform infrared (FTIR) spectroscopy. The data are compared to the structure obtained by X-ray analysis of the crystalline enzyme. The secondary structure was determined using the spectral features observed in the amide-I band (H,O buffer) and amide-I' band (D,O buffer) at 1600-1700 cm-I, taking advantage of resolution-enhancement techniques along with least-squares band-fitting procedures. The relative amounts of different secondary-structure elements, 34-36% for a-helices, 19-25% for p-sheets, 15-16% for turns and 13-17% for irregular structures, were estimated. These data, obtained with the enzyme in solution, correlate well with X-ray data of the crystalline protein [Sussman, J. L., Hard, M., Frolow, F., Oefner, C., Goldman, A., Toker, L. The relationship between the thermally induced loss of enzyme activity and secondary-structure changes has also been investigated. The decrease in enzyme activity to zero at 30-40 "C was accompanied only by minor changes in the secondary structure. At 55-6OoC, denaturation of AChE occurs. In this temperature range, all bands assigned to the various secondary-structure elements abruptly disappear in a co-operative and irreversible manner, whereas the &aggregation bands (at 1622 cm-' and the corresponding high-frequency band) increase in intensity at the same rate.Acetylcholinesterase (AChE) is a key molecule involved in nerve-impulse transmission at cholinergic synapses. It rapidly hydrolyses the neurotransmitter acetylcholine and thus terminates the signal. As the main point of attack of most insecticides and chemical-warfare agents (nerve gases), AChE is of considerable interest. Moreover, structural research on AChFi is relevant because AChE is the best characterized member of the protein family of serine esterases, as defined on the basis of sequence homology (Myers et al., 1988;Krejci et al., 1991;Ollis et al., 1992). Since disulfide bridges are highly conserved within this protein family, it is plausible that AChE shares secondary-structure and tertiarystructure motifs with these related proteins.The three-dimensional structure of crystallized AChEi has been recently reported (Sussman et al., 1991). The AChE monomer is an ellipsoidally shaped alp-protein, with dimensions of 4.3 nm X 6.0 nm X 6.5 nm, which contains a 12-stranded mixed P-sheet surrounded by 14 a-helices. From the X-ray data, it can be concluded that the a-structure content is approximately 32% and that the p-structure content is 15 %. Even with detailed structural information from X-ray crystallography, spectroscopic methods for investigating secondary structure have not lost their attraction. Firstly, spectroscopic methods allow the direct measurement of proteins in solution (in a more native state) and they have considerable potential for exploring conformational changes, such as temperature-dependent processes, which might not, in principle, be amenable to X-ray analysis. Secondly, ...

Fourier Transform Infrared Spectrometric Analysis of Protein Conformation: Effect of Sampling Method and Stress Factors

“…To verify this assignment heat-denatured lyophilized protein aggregates were investigated. It is known that novel bands arise around 1620 -1630 and 1695 cm Ϫ1 in the spectra of protein aggregates in solution (H 2 O) (13,17,31,32). These bands originate from intermolecular antiparallel ␤-sheet formation, and usually the 1620 -1630 cm Ϫ1 band is much more intense.…”

Temperature scanning FTIR analysis of secondary structures of proteins embedded in amorphous sugar matrix

“…The resolution factor, K, and half-width of the unresolved bands were 2.8 and 30 cm À1 , respectively, which are in the range of previously published values. 9,[17][18][19][20] The overlapping bands in the deconvolved amide I band region were resolved using a PeakFit software program (SeaSolve Software Inc., Framingham, MA). The resolved bands were assigned to the secondary structural components, as previously described in the literature.…”

Lyophilization-induced reversible changes in the secondary structure of proteins.

“…Thermal aggregation raises the fl-sheet content in acetylcholinesterase (58) and ovalbumin (59). Precipitation with salt increases the fl-sheet content and decreases the a-helix content of proteins (60).…”