In the past decades, a multitude of enhanced algorithms, based on variable selection, or singular value decomposition of standardized, scaled, and calibrated reference spectra, have been proposed to predict the secondary structure content, with good overall secondary structure prediction ( 5, 6 ).

Louis, MO, and approved May 6, 2015 (received for review January 15, 2015).

However, quantitative estimation for -sheetcontaining proteins is problematic due to the huge morphological and spectral diversity of -structures.

Taking into account the twist of -structures, our method accurately estimates the secondary structure for a broad range of protein folds, particularly for -sheetrich proteins and amyloid fibrils.

Moreover, the method can predict the protein fold down to the topology level following the CATH classification.

We provide a general tool for a quick and reliable structure analysis using conventional or synchrotron radiation CD spectroscopy, which is especially useful when X-ray or NMR techniques fail.

Abstract Circular dichroism (CD) spectroscopy is a widely used technique for the study of protein structure.

Numerous algorithms have been developed for the estimation of the secondary structure composition from the CD spectra.

These methods often fail to provide acceptable results on -mixed or -structurerich proteins.

The problem arises from the spectral diversity of -structures, which has hitherto been considered as an intrinsic limitation of the technique.

The predictions are less reliable for proteins of unusual -structures such as membrane proteins, protein aggregates, and amyloid fibrils.

Here, we show that the parallelantiparallel orientation and the twisting of the -sheets account for the observed spectral diversity.

We have developed a method called -structure selection (BeStSel) for the secondary structure estimation that takes into account the twist of -structures.

This method can reliably distinguish parallel and antiparallel -sheets and accurately estimates the secondary structure for a broad range of proteins.

Moreover, the secondary structure components applied by the method are characteristic to the protein fold, and thus the fold can be predicted to the level of topology in the CATH classification from a single CD spectrum.

By constructing a web server, we offer a general tool for a quick and reliable structure analysis using conventional CD or synchrotron radiation CD (SRCD) spectroscopy for the protein science research community.

The method is especially useful when X-ray or NMR techniques fail.

Using BeStSel on data collected by SRCD spectroscopy, we investigated the structure of amyloid fibrils of various disease-related proteins and peptides.

The far-UV circular dichroism (CD) spectroscopy of proteins and peptides (180250 nm) is predominantly based on the excitation of electronic transitions in amide groups.

The peptide backbone forms characteristic secondary structures such as -helices, -pleated sheets, turns, and disordered sections with specific, dihedral angles and H-bond patterns affecting the CD spectrum ( 1 ).

CD has been exploited for protein folding and stability assays, intermolecular interactions, and ligand binding studies, and has recently been applied in the investigations of protein disorder ( 2, 3 ).

Synchrotron radiation CD (SRCD) spectroscopy is an emerging technique complementary to small-angle X-ray scattering or infrared spectroscopy, synergistic to biochemical and biophysical assays characterizing the protein folding state.

SRCD extends the limits of conventional CD spectroscopy by broadening the spectral range, increasing the signal-to-noise ratio, and accelerating the data acquisition, in the presence of absorbing components (buffers, salts, etc.) ( 4 ).

Additionally, SRCD has the capability of time-resolved and stopped-flow measurements as well as high-throughput screening ( 3 ).

Quantitative analysis of CD spectra allows the prediction of the protein secondary structure content.

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