Improved Estimation of the Secondary Structures of Proteins by Vacuum-Ultraviolet Circular Dichroism Spectroscopy

Abstract

The vacuum-ultraviolet circular dichroism (VUVCD) spectra of 16 globular proteins (insulin, lactate dehydrogenase, glucose isomerase, lipase, conalbumin, transferrin, catalase, subtilisin A, alpha-amylase, staphylococcal nuclease, papain, thioredoxin, carbonic anhydrase, elastase, avidin, and xylanase) were successfully measured in aqueous solutions at 25 degrees C from 260 to 160 nm under a high vacuum using a synchrotron-radiation VUVCD spectrophotometer. These proteins exhibited characteristic CD spectra below 190 nm that were related to their different secondary structures, which could not be detected with a conventional CD spectrophotometer. The component spectra of alpha-helices, beta-strands, turns, and unordered structures were obtained by deconvolution analysis of the VUVCD spectra of 31 reference proteins including the 15 proteins reported in our previous paper [Matsuo, K. et al. (2004) J. Biochem. 135, 405-411]. Prediction of the secondary-structure contents using the SELCON3 program was greatly improved, especially for alpha-helices, by extending the short-wavelength limit of CD spectra to 160 nm and by increasing the number of reference proteins. The numbers of alpha-helix and beta-strand segments, which were calculated from the distorted alpha-helix and beta-strand contents, were close to those obtained on X-ray crystallography. These results demonstrate the usefulness of synchrotron-radiation VUVCD spectroscopy for the secondary structure analysis of proteins.

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.…”

Construction of a Synchrotron-Radiation Vacuum-Ultraviolet Circular-Dichroism Spectrophotometer and Its Application to the Structural Analysis of Biomolecules

“…3436 Figure 11a shows the VUVCD spectra of some typical globular proteins (myoglobin, lysozyme, concanavalin A, and soybean trypsin inhibitor). 37,38 Myoglobin, which is mainly composed of ¡-helix (76%), exhibits three negative peaks around 222, 208, and 170 nm, a positive peak around 190 nm, and a shoulder around 175 nm. The pattern of the spectrum for lysozyme, with ¡-helices (42%) and ¢-strands (6%), is similar to that of myoglobin, but its CD peaks are much smaller.…”

“…Figure 11b shows the component spectra of four secondary structures (¡-helices, ¢-strands, turns, and unordered structures) down to 160 nm, which were extracted from the VUVCD spectra of 31 reference proteins by deconvolution analysis with the SELCON3 program. 38 These reference proteins, which were typically used in the previous database, 34 were selected to have a balanced content of each secondary structure. The VUVCD spectrum of ¡-helix is characterized by a large positive peak around 193 nm and a negative peak around 167 nm, in addition to the two well-known negative peaks around 208 and 220 nm.…”