BeStSel: analysis site for protein CD spectra-2025 update

Abstract

Circular dichroism (CD) spectroscopy is a widely used technique to characterize the secondary structure composition of proteins. We have developed the Beta Structure Selection (BeStSel) method (PNAS, 112, E3095), which solves the main problem of protein CD spectroscopy-namely, the spectral variability of β-structures. The BeStSel web server utilizes this method to provide tools to the community for CD spectrum analysis. BeStSel uniquely provides information on eight secondary structure components, including parallel β-structure and antiparallel β-sheets with three different twist groups. It outperforms all available methods in accuracy and information content, and is also able to predict protein folds down to the topology/homology level of the CATH classification. The algorithm has been further developed, and the accuracy of the estimation of the secondary structure elements is improved by 0.7% as an average on the reference dataset. A new module of the web server calculates protein stability from the thermal denaturation profile followed by CD. Secondary structure calculations of uploaded PDB and mmcif files support the experimental verification of MD simulations and AlphaFold models by CD spectroscopy. Well-proven modules for disorder-order classification and extinction coefficient calculation continue to work. The BeStSel server is freely accessible at https://bestsel.elte.hu.

Graphical Abstract

Figure 1.

Investigation of the structure of α-synuclein by CD spectroscopy and validation of its AlphaFold prediction. (A) AlphaFold2 model of α-synuclein, predicting 64% α-helix structure with high confidence score (represented by blue color). (B) CD spectroscopy proves that the monomer α-synuclein under physiological conditions exhibits a CD spectrum characteristic of disordered structure. Moreover, α-synuclein migh form β-sheet rich oligomers, as well, and can take α-helical conformation in trifluoro-ethanol. These CD spectra were measured on a JASCO J-810 spectropolarimeter (JASCO Co., Tokyo, Japan) and used previously to present the Disordered-Ordered Classification tool of BeStSel [12]. (C) Secondary structure estimation by BeStSel for the α-synuclein forms in comparison to the secondary structure contents of the AlphaFold2 model.

Figure 2.

Validation of the structure of recombinant β₂-microglobulin (β2m) by CD spectroscopy. (A) X-ray structure (2YXF) of β2m consisting of mainly antiparallel β-sheets [72]. (B) Experimental CD spectrum of the protein recorded by SRCD at the DISCO beamline, SOLEIL Synchrotron [3, 11], and CD spectrum calculated in silico for the 2YXF.pdb structure by the SESCA algorithm [70]. Two validation methods are presented here. As the goal is to validate the structure of the recombinant protein, the experimental CD spectrum was analyzed by BeStSel and compared to the secondary structure derived from the X-ray structure by using the “Secondary structure from PDB files” function of the BeStSel web server, showing good agreement (C). Another method is to match the experimental CD to a calculated CD spectrum [70, 71]. We observe significant spectral differences and large RMSD between them (B), which we might interpret so that the structures behind are different, i.e. the measured protein structure is different from the one deposited in the PDB. For curiosity, we analyzed the calculated CD spectrum with BeStSel, and it showed a similar secondary structure, although with some differences, to the X-ray structure and to the results of the experimental data analysis (C).