Secondary-Structure Analysis of Proteins by Vacuum-Ultraviolet Circular Dichroism Spectroscopy

Abstract

The vacuum ultraviolet circular dichroism (VUVCD) spectra of 15 globular proteins (myoglobin, hemoglobin, human serum albumin, cytochrome c, peroxidase, alpha-lactalbumin, lysozyme, ovalbumin, ribonuclease A, beta-lactoglobulin, pepsin, trypsinogen, alpha-chymotrypsinogen, soybean trypsin inhibitor, and concanavalin A) were measured in aqueous solutions at 25 degrees C in the wavelength region from 260 to 160 nm under a high vacuum, using a synchrotron-radiation VUVCD spectrophotometer. The VUVCD spectra below 190 nm revealed some characteristic bands corresponding to different secondary structures. The contents of alpha-helices, beta-strands, turns, and unordered structures were estimated using the SELCON3 program with VUVCD spectra data on the 15 proteins. Prediction of the secondary-structure contents was greatly improved by extending the circular dichroism spectra to 165 nm. The numbers of alpha-helix and beta-strand segments calculated from the distorted alpha-helix and beta-strand contents did not differ greatly from those obtained from X-ray crystal structures. These results demonstrate that synchrotron-radiation VUVCD spectroscopy is a powerful tool for analyzing the secondary structures of proteins.

First‐principles calculations of protein circular dichroism in the far‐ultraviolet and beyond

“…There is still much to be done to assess the quality of matrix method calculations in the vacuum-UV and the extent to which they agree with experimental observations around 165 nm. 58,59 Our results also have some bearing on the deep-UV, as the transitions from the bonding p molecular orbital to the antibonding orbital are calculated to occur around 130 nm. The electric transition dipole moments of the local p nb ?p* and local p b ?p* transitions are perpendicular to one another.…”

“…Several recent studies have indicated the presence of features in the CD spectra of proteins around 165 nm. 58,59 There has been some speculation in the literature over the years as to the nature of the electronic transitions that might lead to these bands around 165 nm. Suggestions have included excitations to an antibonding r orbital, from either the higher-energy lone pair orbital on oxygen, nr*, or from the nonbonding p molecular orbital, pr*.…”

Overexpression, purification, and characterization of Paenibacillus cell surface-expressed chitinase ChiW with two catalytic domains

“…5) because VUVCD spectroscopy is a powerful technique for analyzing the secondary structures of proteins. 32,33) The spectrum exhibited a positive peak around 195 nm and two negative peaks around 220 and 170 nm, indicating that the recombinant ChiW-SLHd folded with some α-helix and β-stranded structures. The α-helix and β-strand contents of ChiW-SLHd estimated by using the SELCON3 program and the VUVCD spectrum down to 168 nm showed 22.8 and 29.1%, respectively.…”

“…The details of the optical devices, sample cell, and data acquisition of the spectrophotometer were described previously. [30][31][32][33] The enzyme (141 μM) was prepared in buffer containing 10 mM sodium phosphate, pH 7.4. The secondary structure contents of ChiW-SLHd were estimated using the SELCON3 program 34) and the VUVCD spectra of the 31 reference proteins with known X-ray structures.…”

“…The secondary structure contents of ChiW-SLHd were estimated using the SELCON3 program 34) and the VUVCD spectra of the 31 reference proteins with known X-ray structures. 32,33) The secondary structures of reference proteins in crystal form were assigned using the DSSP program.…”