Glycerol-Induced Folding of Unstructured Disulfide-Deficient Lysozyme into a Native-Like Conformation

摘要

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 front 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, H-1-N-15-HSQC spectra of partially deuterated Protei. it 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 beta 3-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.