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 α/β-mixed or β-structure-rich proteins. The problem arises from the spectral diversity of β-structures, which has hitherto been considered as an intrinsic limitation of the technique. The predictions are less reliable for proteins of unusual β-structures such as membrane proteins, protein aggregates, and amyloid fibrils. Here, we show that the parallel/antiparallel orientation and the twisting of the β-sheets account for the observed spectral diversity. We have developed a method called β-structure selection (BeStSel) for the secondary structure estimation that takes into account the twist of β-structures. This method can reliably distinguish parallel and antiparallel β-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. circular dichroism | secondary structure determination | protein fold | protein aggregation | amyloid O ptically active macromolecules, such as proteins, exhibit differential absorption of circular polarized light. The far-UV circular dichroism (CD) spectroscopy of proteins and peptides (180-250 nm) is predominantly based on the excitation of electronic transitions in amide groups. The peptide backbone forms characteristic secondary structures such as α-helices, β-pleated sheets, turns, and disordered sections with specific Φ, Ψ 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 hig...

Construction of the presynaptic filament (PSF) of proper helical structure by Rad51 recombinases is a prerequisite of the progress of homologous recombination repair. We studied the contribution of ATP-binding to this structure of wt human Rad51 (hRad51). We exploited the protein-dissociation effect of high hydrostatic pressure to determine the free energy of dissociation of the protomer interfaces in hRad51 oligomer states and used electron microscopy to obtain topological parameters. Without cofactors ATP and Ca(2+) and template DNA, hRad51 did not exist in monomer form, but it formed rodlike long filaments without helical order. ΔG(diss) indicated a strong inherent tendency of aggregation. Binding solely ssDNA left the filament unstructured with slightly increased ΔG(diss). Adding only ATP and Ca(2+) to the buffer disintegrated the self-associated rods into rings and short helices of further increased ΔG(diss). Rad51 binding to ssDNA only with ATP and Ca bound could lead to ordered helical filament formation of proper pitch size with interface contacts of K(d) ∼ 2 × 10(-11) M, indicating a structure of outstanding stability. ATP/Ca binding increased the ΔG(diss) of protomer contacts in the filament by 16 kJ/mol. The results emphasize that ATP-binding in the PSF of hRad51 has an essential, yet purely structural, role.