An experimental study of GFP‐based FRET, with application to intrinsically unstructured proteins

Abstract

We have experimentally studied the fluorescence resonance energy transfer (FRET) between green fluorescent protein (GFP) molecules by inserting folded or intrinsically unstructured proteins between CyPet and Ypet. We discovered that most of the enhanced FRET signal previously reported for this pair was due to enhanced dimerization, so we engineered a monomerizing mutation into each. An insert containing a single fibronectin type III domain (3.7 nm end-to-end) gave a moderate FRET signal while a two-domain insert (7.0 nm) gave no FRET. We then tested unstructured proteins of various lengths, including the charged-plus-PQ domain of ZipA, the tail domain of a-adducin, and the C-terminal tail domain of FtsZ. The structures of these FRET constructs were also studied by electron microscopy and sedimentation. A 12 amino acid linker and the N-terminal 33 amino acids of the charged domain of the ZipA gave strong FRET signals. The C-terminal 33 amino acids of the PQ domain of the ZipA and several unstructured proteins with 66-68 amino acids gave moderate FRET signals. The 150 amino acid charged-plus-PQ construct gave a barely detectable FRET signal. FRET efficiency was calculated from the decreased donor emission to estimate the distance between donor and acceptor. The donor-acceptor distance varied for unstructured inserts of the same length, suggesting that they had variable stiffness (persistence length). We conclude that GFP-based FRET can be useful for studying intrinsically unstructured proteins, and we present a range of calibrated protein inserts to experimentally determine the distances that can be studied.

Structural rearrangement of the intracellular domains during AMPA receptor activation

“…There are 39 amino acids between the end of M4 (Lys-848) and the I10 FP insert (after Asn-864 and including the 13-residue unstructured linker to the N terminus of the FP). Flexible polypeptides of 50 residues connecting FPs exhibit similar FRET distances (∼60 Å) (42), which includes a minimum 20-Å separation due to volume exclusion from the size of the two fluorophores. Thus, although the C terminus might contain secondary structure elements, it must be largely disordered.…”

Weak protein–protein interactions in live cells are quantified by cell-volume modulation

“…Moving on to putative quinary interactions, we first examine the association of commonly used FP labels. Wild-type FPs form homo-oligomers (42,43) and have a high degree of sequence and structural homology, making heterooligomerization, even of engineered FPs, a realistic possibility. Fluorescent protein heterooligomerization presents problems in the interpretation of FRET and colocalization experiments, yet has scarcely been studied (44).…”

Identification and Functional Impact of Homo-oligomers of the Human Proton-coupled Folate Transporter

“…To prepare fluorescent protein-tagged hPCFTs for the fluorescence resonance energy transfer (FRET) experiments (see below), YPet-His and ECFP*-His cDNAs optimized for mammalian codon expression of yellow and enhanced cyan fluorescent proteins, respectively, were provided by Dr. Tomoo Ohashi (Duke University Medical Center) (43). To generate monomeric YPet (mYPet) and ECFP* (mECFP*), Ala-206 was substituted with lysine in both constructs, as described for other GFP variants (44).…”