Domain-specific fluorescence resonance energy transfer (FRET) sensors of metallothionein/thionein.
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.
Hong
S-H
SH
Center for Biochemical and Biophysical Sciences and Medicine, Department of Pathology, Harvard Medical School, One Kendall Square, Cambridge, MA 02139, USA.
Hao
Q
Q
Maret
W
W
D015374:Biosensing Techniques; D003545:Cysteine / Q000737:chemistry; D004880:Ergothioneine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D008668:Metallothionein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D009282:Naphthalenesulfonates / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D020033:Protein Isoforms; D011994:Recombinant Proteins / Q000737:chemistry; D013439:Sulfhydryl Reagents / Q000737:chemistry / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism*; D015032:Zinc / Q000737:chemistry
D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D020033:Protein Isoforms; D011994:Recombinant Proteins; D013439:Sulfhydryl Reagents; C006421:1,5-I-AEDANS; D014364:Tryptophan; D008668:Metallothionein; D004880:Ergothioneine; D015032:Zinc; D003545:Cysteine