Abstract
Far-UV circular dichroism (CD) spectroscopy is a classical method for the study of the secondary structure of polypeptides in solution. It has been the general view that the α-helix content can be estimated accurately from the CD spectra. However, the technique was less reliable to estimate the β-sheet contents as a consequence of the structural variety of the β-sheets, which is reflected in a large spectral diversity of the CD spectra of proteins containing this secondary structure component. By taking into account the parallel or antiparallel orientation and the twist of the β-sheets, the Beta Structure Selection (BeStSel) method provides an improved β-structure determination and its performance is more accurate for any of the secondary structure types compared to previous CD spectrum analysis algorithms. Moreover, BeStSel provides extra information on the orientation and twist of the β-sheets which is sufficient for the prediction of the protein fold.The advantage of CD spectroscopy is that it is a fast and inexpensive technique with easy data processing which can be used in a wide protein concentration range and under various buffer conditions. It is especially useful when the atomic resolution structure is not available, such as the case of protein aggregates, membrane proteins or natively disordered chains, for studying conformational transitions, testing the effect of the environmental conditions on the protein structure, for verifying the correct fold of recombinant proteins in every scientific fields working on proteins from basic protein science to biotechnology and pharmaceutical industry. Here, we provide a brief step-by-step guide to record the CD spectra of proteins and their analysis with the BeStSel method.
Highlights
Circular dichroism (CD) corresponds to the differential absorption between left and right circularly polarized light (Fig. 1)
Depending on the local geometry, environment, and H-bond pattern of the peptide bonds, the polypeptide chains with different conformations can exhibit distinct, characteristic spectral profiles, which is manifested in the CD spectra of proteins
In the last 30 years, a dozen CD spectrum analysis algorithms made attempts to accurately estimate the secondary structure composition of the proteins. These methods use reference CD spectra of proteins with known structure to make an estimation of different types of secondary structure elements
Summary
Circular dichroism (CD) corresponds to the differential absorption between left and right circularly polarized light (Fig. 1). 176 Andras Micsonai et al. Recently, we have shown that the spectral contribution of β-sheets depends on the parallel-antiparallel orientation and the twist of the β-sheets [4]. We have shown that the spectral contribution of β-sheets depends on the parallel-antiparallel orientation and the twist of the β-sheets [4] Based on this observation, we have developed a new method named BeStSel (Beta Structure Selection) for the secondary structure estimation of proteins from the CD spectra that takes into account the orientation and twist of the β-sheets. The method defines eight structural components: regular and distorted α-helices, left-handed, relaxed (slightly right-hand twisted) and right-hand twisted antiparallel β-sheets, parallel β-sheet, turn. As a results of the detailed structural information gained from the CD spectrum, BeStSel is capable of predicting the protein fold down to the homology level using the CATH fold classification (Fig. 4) [9, 10]
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