Glycerol-induced folding of unstructured disulfide-deficient lysozyme into a native-like conformation.
2SS[6-127,64-80] variant of lysozyme which has two disulfide bridges, Cys6-Cys127 and Cys64-Cys80, and lacks the other two disulfide bridges, Cys30-Cys115 and Cys76-Cys94, was quite unstructured in water, but a part of the polypeptide chain was gradually frozen into a native-like conformation with increasing glycerol concentration. It was monitored from the protection factors of amide hydrogens against H/D exchange. In solution containing various concentrations of glycerol, H/D exchange reactions were carried out at pH* 3.0 and 4 degrees C. Then, (1)H-(15)N-HSQC spectra of partially deuterated protein were measured in a quenching buffer for H/D exchange (95% DMSO/5% D(2)O mixture at pH* 5.5 adjusted with dichloroacetate). In a solution of 10% glycerol, the protection factors were nearly equal to 10 at most of residues. With increasing glycerol concentration, some selected regions were further protected, and their protection factors reached about a 1000 in 30% glycerol solution. The highly protected residues were included in A-, B-, and C-helices and beta3-strand, and especially centered on Ile 55 and Leu 56. In 2SS[6-127,64-80], long-range interactions were recovered due to the preferential hydration by glycerol in the hydrophobic box of the alpha-domain. Glycerol-induced recovering of the native-like structure is discussed from the viewpoint of molten globules growing with the protein folding. (c) 2009 Wiley Periodicals, Inc. Biopolymers 91: 665-675, 2009.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.
Sakamoto
Keiko
K
School of Science and Technology, Kwansei Gakuin University, Sanda, 669-1337, Japan.
Hirai
Ken-Ichi
K
Kitamura
Yoshiaki
Y
Yamazaki
Kouta
K
Yusa
Mitsunobu
M
Tokunaga
Naoki
N
Doi
Gakuji
G
Noda
Yasuo
Y
Tachibana
Hideki
H
Segawa
Shin-Ichi
S
D000818:Animals; D002645:Chickens; D017666:Deuterium Oxide; D004220:Disulfides / Q000737:chemistry; D014644:Genetic Variation; D005990:Glycerol / Q000494:pharmacology; D066298:In Vitro Techniques; D008958:Models, Molecular; D009113:Muramidase / Q000737:chemistry* / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation / Q000187:drug effects; D017510:Protein Folding / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D004220:Disulfides; D011994:Recombinant Proteins; D009113:Muramidase; D017666:Deuterium Oxide; D005990:Glycerol