title: Secondary-structure analysis of alcohol-denatured proteins by vacuum-ultraviolet circular dichroism spectroscopy.
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.
authors.affiliation: Hiroshima Synchrotron Radiation Center, Hiroshima University, Higashi-Hiroshima, Japan.
authors.forename: Koichi
authors.initials: K
authors.lastname: Matsuo
authors.forename: Yoshie
authors.initials: Y
authors.lastname: Sakurada
authors.forename: Shin-ichi
authors.initials: S
authors.lastname: Tate
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Namatame
authors.forename: Masaki
authors.initials: M
authors.lastname: Taniguchi
authors.forename: Kunihiko
authors.initials: K
authors.lastname: Gekko
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002919:Chymotrypsinogen / Q000737:chemistry; D002942:Circular Dichroism; D003198:Computer Simulation; D029968:Escherichia coli Proteins / Q000737:chemistry; D006736:Horses; D006801:Humans; D007768:Lactalbumin / Q000737:chemistry; D007782:Lactoglobulins / Q000737:chemistry; D000432:Methanol / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009211:Myoglobin / Q000737:chemistry; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D012709:Serum Albumin / Q000737:chemistry; D013879:Thioredoxins / Q000737:chemistry; D014270:Trifluoroethanol / Q000737:chemistry
chemical_list: D029968:Escherichia coli Proteins; D007782:Lactoglobulins; D009211:Myoglobin; D012709:Serum Albumin; D013879:Thioredoxins; D014270:Trifluoroethanol; D007768:Lactalbumin; D002919:Chymotrypsinogen; D000432:Methanol