Glycerol-induced folding of unstructured disulfide-deficient lysozyme into a native-like conformation

• Published in: Biopolymers 2009-08, Vol.91 (8), p.665-675
• Main Authors: Sakamoto, Keiko, Hirai, Ken-ichi, Kitamura, Yoshiaki, Yamazaki, Kouta, Yusa, Mitsunobu, Tokunaga, Naoki, Doi, Gakuji, Noda, Yasuo, Tachibana, Hideki, Segawa, Shin-ichi
• Format: Article
• Language: English
• Subjects: Animals; Chickens; Deuterium Oxide; disulfide-deficient lysozyme; Disulfides - chemistry; Genetic Variation; Glycerol - pharmacology; glycerol-induced folding; H/D exchange; In Vitro Techniques; Models, Molecular; Muramidase - chemistry; Muramidase - genetics; NMR study of unstructured protein; Nuclear Magnetic Resonance, Biomolecular; preferential hydration by glycerol; Protein Conformation - drug effects; Protein Folding - drug effects; Recombinant Proteins - chemistry; Recombinant Proteins - genetics
• ISSN: 0006-3525; 1097-0282
• DOI: 10.1002/bip.21198

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°C. Then, 1H‐15N‐HSQC spectra of partially deuterated protein were measured in a quenching buffer for H/D exchange (95% DMSO/5% D2O 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 β3‐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 α‐domain. Glycerol‐induced recovering of the native‐like structure is discussed from the viewpoint of molten globules growing with the protein folding. © 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 [e-mail]