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Reinvestigation of the oxidative folding pathways of hen egg white lysozyme: switching of the major pathways by temperature control.

ABSTRACT: It has been well established that in the oxidative folding of hen egg white lysozyme (HEL), which has four SS linkages in the native state (N), three des intermediates, i.e., des[76-94], des[64-80], and des [6-127], are populated at 20 °C and N is dominantly formed by the oxidation of des[64-80] and des[6-127]. To elucidate the temperature effects, the oxidative folding pathways of HEL were reinvestigated at 5-45 °C in the presence of 2 M urea at pH 8.0 by using a selenoxide reagent, DHSox. When reduced HEL was reacted with 1-4 equivalents of DHSox, 1S, 2S, 3S, and 4S intermediate ensembles with 1-4 SS linkages, respectively, were produced within 1 min. After the oxidation, 3S was slowly converted to the des intermediates with formation of the native structures through SS rearrangement. At 5 °C, des[76-94] was populated in the largest amount, but the oxidation to N was slower than that of des[64-80] and des[6-127]. At 35 °C, on the other hand, des[64-80] and des[6-127] were no longer stable, and only des[76-94] was populated. The results suggested that the major folding pathways of HEL can be switched from one to the other by temperature control.

SUBMITTER: Arai K

PROVIDER: S-EPMC3742182 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Reinvestigation of the oxidative folding pathways of hen egg white lysozyme: switching of the major pathways by temperature control.

Arai Kenta K Shibagaki Wataru W Shinozaki Reina R Iwaoka Michio M

International journal of molecular sciences 20130626 7

It has been well established that in the oxidative folding of hen egg white lysozyme (HEL), which has four SS linkages in the native state (N), three des intermediates, i.e., des[76-94], des[64-80], and des [6-127], are populated at 20 °C and N is dominantly formed by the oxidation of des[64-80] and des[6-127]. To elucidate the temperature effects, the oxidative folding pathways of HEL were reinvestigated at 5-45 °C in the presence of 2 M urea at pH 8.0 by using a selenoxide reagent, DHSox. When ...[more]

PMID: 23803654

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Project description:Chemical bonding at the active site of hen egg-white lysozyme (HEWL) is analyzed on the basis of Bader's quantum theory of atoms in molecules [QTAIM; Bader (1994), Atoms in Molecules: A Quantum Theory. Oxford University Press] applied to electron-density maps derived from a multipole model. The observation is made that the atomic displacement parameters (ADPs) of HEWL at a temperature of 100?K are larger than ADPs in crystals of small biological molecules at 298?K. This feature shows that the ADPs in the cold crystals of HEWL reflect frozen-in disorder rather than thermal vibrations of the atoms. Directly generalizing the results of multipole studies on small-molecule crystals, the important consequence for electron-density analysis of protein crystals is that multipole parameters cannot be independently varied in a meaningful way in structure refinements. Instead, a multipole model for HEWL has been developed by refinement of atomic coordinates and ADPs against the X-ray diffraction data of Wang and coworkers [Wang et al. (2007), Acta Cryst. D63, 1254-1268], while multipole parameters were fixed to the values for transferable multipole parameters from the ELMAM2 database [Domagala et al. (2012), Acta Cryst. A68, 337-351] . Static and dynamic electron densities based on this multipole model are presented. Analysis of their topological properties according to the QTAIM shows that the covalent bonds possess similar properties to the covalent bonds of small molecules. Hydrogen bonds of intermediate strength are identified for the Glu35 and Asp52 residues, which are considered to be essential parts of the active site of HEWL. Furthermore, a series of weak C-H...O hydrogen bonds are identified by means of the existence of bond critical points (BCPs) in the multipole electron density. It is proposed that these weak interactions might be important for defining the tertiary structure and activity of HEWL. The deprotonated state of Glu35 prevents a distinction between the Phillips and Koshland mechanisms.

Dataset Information

Reinvestigation of the oxidative folding pathways of hen egg white lysozyme: switching of the major pathways by temperature control.

Publications

Reinvestigation of the oxidative folding pathways of hen egg white lysozyme: switching of the major pathways by temperature control.

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