Synergistic effects of copper sites on apparent stability of multicopper oxidase, Fet3p
Journal article, 2018

Saccharomyces cerevisiae Fet3p is a multicopper oxidase that contains three cupredoxin-like domains and four copper ions located in three distinct metal sites (T1 in domain 3; T2 and the binuclear T3 at the interface between domains 1 and 3). To probe the role of the copper sites in Fet3p thermodynamic stability, we performed urea-induced unfolding experiments with holo-, apoand three partially-metallated (T1, T2 and T1/T2 sites depleted of copper) forms of Fet3p. Using a combination of spectroscopic probes (circular dichroism, fluorescence intensity and maximum, 8-anilinonaphthalene-1-sulfonic acid (ANS) emission, oxidase activity and blue color), we reveal that all forms of Fet3p unfold in a four-state reaction with two partially-folded intermediates. Using phase diagrams, it emerged that Fet3p with all copper sites filled had a significantly higher stability as compared to the combined contributions of the individual copper sites. Hence, there is long-range inter-domain communication between distal copper sites that contribute to overall Fet3p stability.

Phase diagram method

Multicopper oxidases MCO

Multidomain protein stability

Cupredoxin-like domain

Fet3p

Author

Erik Sedlák

Pavol Jozef Safarik University

Gabriel Žoldák

Pavol Jozef Safarik University

Pernilla Wittung Stafshede

Chalmers, Biology and Biological Engineering, Chemical Biology

International Journal of Molecular Sciences

1422-0067 (ISSN)

Vol. 19 1 269

Subject Categories

Biochemistry and Molecular Biology

Other Basic Medicine

Structural Biology

DOI

10.3390/ijms19010269

More information

Latest update

4/20/2018