title: An electrochemical method for investigation of conformational flexibility of active sites of Trametes versicolor laccase based on sensitive determination of copper ion with cysteine-modified electrodes.
abstract: This study demonstrates a facile yet effective electrochemical method to investigate the conformational flexibility of the active sites of Trametes versicolor (Tv) laccase based on sensitive determination of copper ions (Cu(2+)) dissociated from the enzyme with the cysteine-modified Au electrodes. In the native state, the multicopper active sites are deeply buried in the polypeptide of Tv laccase and are thus not electrochemically detectable even at the cysteine-modified Au electrodes. Upon the unfolding of Tv laccase induced by guanidine hydrochloride (GdnHCl), copper ions dissociate from the peptide chain and, as a consequence, are electrochemically reduced and thus detected at the cysteine-modified Au electrodes. Such a property could be used to investigate the conformational flexibility of multicopper active sites of Tv laccase in a simple way. We find that both the conformation of the multicopper active sites in Tv laccase and the enzyme activity change with the presence of a low concentration of GdnHCl denaturant (midpoint, where 50% of the enzyme is unfolded, at 0.7 M). This concentration is lower than that required to induce the conformational changes of Tv laccase molecule as a whole (midpoint at 3.4 M), as investigated by the intrinsic fluorescence of Tv laccase. This observation suggests that the multicopper active sites are formed by relatively weak interactions and hence may be conformationally more flexible than the intact enzyme. The electrochemical method demonstrated in this study is technically simple yet effective and could be potentially useful for investigation on the thermodynamics and kinetics of the conformational changes of multicopper oxidases induced by different denaturants.
authors.affiliation: Beijing National Laboratory for Molecular Sciences, Institute of Chemistry, the Chinese Academy of Sciences (CAS), Beijing 100190, China.
authors.forename: Xianchan
authors.initials: X
authors.lastname: Li
authors.forename: Ping
authors.initials: P
authors.lastname: Yu
authors.forename: Lifen
authors.initials: L
authors.lastname: Yang
authors.forename: Fuyi
authors.initials: F
authors.lastname: Wang
authors.forename: Lanqun
authors.initials: L
authors.lastname: Mao
mesh_terms: D020134:Catalytic Domain; D003300:Copper / Q000032:analysis* / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry*; D004563:Electrochemistry / Q000295:instrumentation*; D004566:Electrodes; D006046:Gold / Q000737:chemistry; D019791:Guanidine / Q000494:pharmacology; D042845:Laccase / Q000737:chemistry*; D058767:Protein Unfolding / Q000187:drug effects; D055454:Trametes / Q000201:enzymology*
chemical_list: D006046:Gold; D003300:Copper; D042845:Laccase; D019791:Guanidine; D003545:Cysteine