Structural and functional alterations of two multidomain oxidoreductases induced by guanidine hydrochloride

Abstract

The unfolding and refolding of two multidomain oxidoreductases, bovine liver catalase and flavoprotein bovine milk xanthine oxidase (XO), have been analyzed by fluorescence spectroscopy, circular dichroism, and activity measurements. Two intermediates, a partially folded active dimer disassembled from the native tetramer and a partially folded inactivated monomer, are found to exist in the conformational changes of catalase induced by guanidine hydrochloride (GdnHCl). Similarly, two intermediates, an active, compacted intermediate bound by flavin adenine dinucleotide (FAD) partially and an inactive flexible intermediate with FAD completely dissociated, exist in the conformational changes of XO induced by GdnHCl. The activity regains completely and an enhancement in activity compared with the native catalase or native XO is observed by dilution of catalase or XO incubated with GdnHCl at concentrations not >0.5 or 1.8 M into the refolding buffer, but the yield of reactivation for catalase or XO is zero when the concentration of GdnHCl is >1.5 or 3.0 M. The addition of FAD provides a remarkable protection against the inactivation of XO by GdnHCl under mild denaturing conditions, and the conformational change of XO is irreversible after FAD has been removed in the presence of a strong denaturing agent. These findings provide impetus for exploring the influences of cofactors such as FAD on the structure-function relationship of xanthine oxidoreductases.

Analysis of the Thermally Induced Structural Changes of Bovine Lactoferrin

“…The linear dependence of I 320 versus I 365 gives indications about the all-or-none transition between two different conformations . On the other hand, the nonlinearity of this function reflects the sequential character of structural transformations . Moreover, each linear portion will describe an individual all-or-none transition.…”

“…35 On the other hand, the nonlinearity of this function reflects the sequential character of structural transformations. 36 Moreover, each linear portion will describe an individual all-or-none transition. This method was employed here to detect the existence of intermediate folding states of LF.…”

Probing the heat‐induced structural changes in bovine serum albumin by fluorescence spectroscopy and molecular modelling

“…The phase diagram was nonlinear, indicating BSA sequential structural heat‐induced changes (Jiao et al . ), involving an unfolding step at temperatures ranging from 25 °C to 40 °C, followed by folding and aggregation at higher temperatures. Dong et al .…”

“…The fluorescence phase diagram describing the heat-induced structural changes in BSA at the different temperatures is shown in Figure 2. The phase diagram was nonlinear, indicating BSA sequential structural heat-induced changes (Jiao et al 2010), involving an unfolding step at temperatures ranging from 25°C to 40°C, followed by folding and aggregation at higher temperatures. Dong et al (2000) suggested that thermal aggregation of proteins is usually characterised as an irreversible two-state model.…”

Probing thermal behaviour of microbial transglutaminase with fluorescence and in silico methods

“…The linear dependence of I 320 versus I 365 gives indications about the all‐or‐none transition between two different conformations . On the other hand, the non‐linearity of this function reflects the sequential character of structural transformations . Moreover, each linear portion will describe an individual all‐or‐none transition.…”