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PEDS Advance Access originally published online on May 23, 2005
Protein Engineering Design and Selection 2005 18(6):255-263; doi:10.1093/protein/gzi031
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oupjournals.org

Domain-specific fluorescence resonance energy transfer (FRET) sensors of metallothionein/thionein

S.-H. Hong1,2, Q. Hao3 and W. Maret1,3,4

1Center for Biochemical and Biophysical Sciences and Medicine, Department of Pathology, Harvard Medical School, One Kendall Square, Cambridge, MA 02139 and 3Departments of Preventive Medicine and Community Health and Anesthesiology, The University of Texas Medical Branch, 700 Harborside Drive, Galveston, TX 77555-1109, USA 2Present address: Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada V6T 1Z3

4 To whom correspondence should be addressed at the Department of Preventive Medicine and Community Health, The University of Texas Medical Branch E-mail: womaret{at}utmb.edu

Each of the two domains of mammalian metallothioneins contains a zinc–thiolate cluster. Employing site-directed mutagenesis and chemical modification, fluorescent probes were introduced into human metallothionein (isoform 2) with minimal perturbations of the structures of these clusters. The resulting FRET (fluorescence resonance energy transfer) sensors are specific for each domain. The design and construction of a sensor for the {alpha}-domain cluster is based on a FRET pair where a C-terminally added tryptophan serves as the donor for a fluorescence acceptor attached to a free cysteine in the linker region between the two domains. Molecular modeling studies and steady-state fluorescence polarization anisotropy measurements suggest unrestricted motion of the tryptophan donor, but limited motion of the AEDANS ({[(amino)ethyl]amino}naphthalene-1-sulfonic acid) acceptor, putting constraints on the use of the {alpha}-domain sensor with this FRET pair as a spectroscopic ruler. The fluorescent metallothioneins allow distance measurements during binding and removal of metals in the individual domains. The overall dimensions of the apoprotein, thionein, for which no structural information is available, do not seem to be significantly different from those of the holoprotein. The single- and double-labeled fluorescent metallothioneins overcome a longstanding impediment in studies of the function of this protein, namely its lack of intrinsic probe characteristics.

Keywords: fluorescent probes/FRET sensor/metallothionein/site-directed mutagenesis/zinc–thiolate clusters

Received August 9, 2004; revised March 1, 2005; accepted April 22, 2005.

Edited by Harold Scheraga


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