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PEDS Advance Access published online on May 23, 2005
Protein Engineering Design and Selection, 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
Received August 9, 2004
Revised March 1, 2005
Accepted April 22, 2005

Article

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

S.-H. Hong 1, Q. Hao 2, and W. Maret 3*

1 Center for Biochemical and Biophysical Sciences and Medicine, Department of Pathology, Harvard Medical School, One Kendall Square, Cambridge, MA 02139
2 Department of Preventive Medicine, The University of Texas Medical Branch, 700 Harborside Drive, Galveston, TX 77555-1109, USA; Department of Community Health, The University of Texas Medical Branch, 700 Harborside Drive, Galveston, TX 77555-1109, USA; Department of Anesthesiology, The University of Texas Medical Branch, 700 Harborside Drive, Galveston, TX 77555-1109, USA
3 Center for Biochemical and Biophysical Sciences and Medicine, Department of Pathology, Harvard Medical School, One Kendall Square, Cambridge, MA 02139; Department of Preventive Medicine, The University of Texas Medical Branch, 700 Harborside Drive, Galveston, TX 77555-1109, USA; Department of Community Health, The University of Texas Medical Branch, 700 Harborside Drive, Galveston, TX 77555-1109, USA; Department of Anesthesiology, The University of Texas Medical Branch, 700 Harborside Drive, Galveston, TX 77555-1109, USA

* To whom correspondence should be addressed.
W. Maret, E-mail: womaret{at}utmb.edu


  Abstract

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.
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