Determination of protein secondary structure in solution by vacuum ultraviolet circular dichroism

Abstract

A new approach is presented for evaluating the secondary structure of globular proteins in solution by analysis of circular dichroism spectra. Two major improvements are introduced. First, extension of the ultraviolet region down to 165 nm. Extension of the spectral region to the vacuum ultraviolet allows observation of new characteristic bands, which improves substantially the analysis of protein conformation. Secondly, new reference spectra that contain all main protein conformations, including two sets of (β-turns, are taken into account. One set of β-turns representative of all main types of turns, contains the sequences (Ala 2-Gly 2) n, l-Pro- d-Ala, N-acetyl- l-Pro-Gly- l-Leu-OH. A comparison of the results of the analysis with one β-turn and with a set of turns indicates that there is essentially no major improvement; thus one β-turn can be taken as the basis spectrum. In addition, the reference spectra include the β-form represented by polypeptide chains composed of alternating hydrophobic and hydrophilic amino acids, which is more closely related to anti-parallel β-pleated sheet structures in proteins. It appears possible to determine the content of antiparallel and of parallel β-pleated sheets with the present basic β-spectrum. The circular dichroism of aromatic side-chain chromophores was measured in the vacuum ultraviolet in order to take their contribution into account. It appears that for proteins with higher aromatic amino acid content and with weak circular dichroism amide bands, it is necessary to take into account a correction for the aromatic side-chain contribution. The conformation of 13 proteins representative of all main classes of globular proteins was determined from circular dichroism data between 165 nm and 250 nm at 0.2 nm intervals. In particular, the secondary structure of the β-rich class of proteins (concanavalin A, Bence-Jones protein V REI, prealbumin, superoxide dismutase), the α/β class of proteins (lactate dehydrogenase, triose phosphate isomerase, subtilisin, glyceraldehyde-3-phosphate dehydrogenase, flavodoxin and pyruvate kinase) and the (α + β) class of proteins (lysozyme and cytochrome c) were quantitatively characterized in solution. The reliability of the method was also assessed by comparing the data obtained with those available from X-ray crystallography. In general, a good agreement was found between the present circular dichroism data and published X-ray data, except for rubredoxin. By including all types of secondary structure and by enlarging substantially the spectral region, our method permits analysis of the conformation of a variety of proteins, such as nucleic acid-binding proteins, immunoglobulins and β-pleated sheet-rich proteins. It offers new possibilities for investigating the dynamics of protein conformation in solution.