Secondary-structure analysis of alcohol-denatured proteins by vacuum-ultraviolet circular dichroism spectroscopy

✍ Scribed by Koichi Matsuo; Yoshie Sakurada; Shin-ichi Tate; Hirofumi Namatame; Masaki Taniguchi; Kunihiko Gekko

• Publisher: John Wiley and Sons
• Year: 2011
• Tongue: English
• Weight: 1009 KB
• Volume: 80
• Category: Article
• ISSN: 0887-3585
• DOI: 10.1002/prot.23206

✦ Synopsis

Abstract

To elucidate the structural characteristics of alcohol‐denatured proteins, we measured the vacuum‐ultraviolet circular dichroism (VUVCD) spectra of six proteins—myoglobin, human serum albumin, α‐lactalbumin, thioredoxin, β‐lactoglobulin, and α‐chymotrypsinogen A—down to 170 nm in trifluoroethanol solutions (TFE: 0−50%) and down to 175 nm in methanol solutions (MeOH: 0−70%) at pH 2.0 and 25°C, using a synchrotron‐radiation VUVCD spectrophotometer. The contents of α‐helices, β‐strands, turns, poly‐L‐proline type II helices (PPIIs), and unordered structures of these proteins were estimated using the SELCON3 program, including the numbers of α‐helix and β‐strand segments. Furthermore, the positions of α‐helices and β‐strands on amino acid sequences were predicted by combining these secondary‐structure data with a neural‐network method. All alcohol‐denatured proteins showed higher α‐helix contents (up to ∼ 90%) compared with the native states, and they consisted of several long helical segments. The helix‐forming ability was higher in TFE than in MeOH, whereas small amounts of β‐strands without sheets were formed in the MeOH solution. The produced α‐helices were transformed dominantly from the β‐strands and unordered structures, and slightly from the turns. The content and mean length of α‐helix segments decreased as the number of disulfide bonds in the proteins increased, suggesting that disulfide bonds suppress helix formation by alcohols. These results demonstrate that alcohol‐denatured proteins constitute an ensemble of many long α‐helices, a few β‐strands and PPIIs, turns, and unordered structures, depending on the types of proteins and alcohols involved. Proteins 2012;. © 2011 Wiley Periodicals, Inc.