Engineering a terbium-binding site into an integral membrane protein for luminescence energy transfer

Abstract

One powerful technique that has not been exploited for studying LacY is f luorescence resonance energy transfer (FRET) (13). This sensitive spectroscopic technique yields information regarding inter-and intramolecular distances. Essentially the method analyzes how the lifetime and quantum yield of a fluorescent molecule (the donor) is influenced by another fluorescent molecule (the acceptor) and vice versa. By using Förster theory, which states that the efficiency of energy transfer is inversely proportional to the sixth power of distance, distances between two fluorophors can be measured (14). The sensitivity and range of distances that can be calculated reliably from FRET depends on the spectral characteristics of the dyes and their relative orientations, which together determine R 0 , the distance at which energy transfer efficiency is 50%. The technique is typically most sensitive for distances between 20 and 100 Å (14, 15).Luminescence resonance energy transfer (LRET) is a particular type of FRET where a lanthanide atom (Tb 3ϩ or Eu 3ϩ ) transfers energy to an organic fluorescent acceptor. The technique exploits the remarkable luminescence properties of lanthanides (i.e., millisecond to submillisecond lifetimes, narrow and multiple emission bands in the visible spectrum, and unpolarized emission). As a consequence, lanthanide luminescence overcomes some of the limitations of conventional FRET experiments [e.g., the unpolarized long lifetime allows random orientation of both the donor and acceptor during the energy transfer process, thereby simplifying determination of the orientation factor between donor and acceptor ( 2 )] (14).Current LRET studies use a thiol-reactive chelator to bind a Tb 3ϩ ion at an engineered Cys residue in the protein (16)(17)(18)(19)(20). The chelator binds Tb 3ϩ with high affinity, shielding the cation from nonradiation deexcitation processes (primarily solvent quenching) (19,21). The resulting Tb 3ϩ emission has a high quantum yield, sometimes approaching unity, and a long lifetime. Although these long-lived lanthanide chelates have been used successfully for estimating inter-and intramolecular distances in the range of 45 Å or more (16-20), they are problematic for short distance measurements in the range desired for most intramolecular interactions because: (i) reactive and highly luminescent chelates are not usually available commercially and require synthesis (21); (ii) the bulk of the chelator may perturb the local environment of the protein; (iii) the mobility inherent in the use of a bulky group with a linker makes distance calculations less precise, particularly under 40 Å; and (iv) both the chelator and the fluorophor are usually directed toward Cys residues, sample heterogeneity is introduced.An alternative possibility for binding Tb 3ϩ at a defined position within a protein is to introduce an EF-hand motif, which Abbreviations: LacY, lactose permease; FRET, fluorescence resonance energy transfer; R0, the distance at which energy transfer efficiency is 50%; LRET, lumines...

Paramagnetic viral nanoparticles as potential high‐relaxivity magnetic resonance contrast agents

“…An equilibrium dissociation constant for Gd 3ϩ binding to CCMV was determined as 31 M by analyzing the decrease in Tb 3ϩ fluorescence intensity upon addition of Gd 3ϩ ( Fig. 4b) (30); the K d Gd 3ϩ in the presence of 1.5 mM Ca 2ϩ was 32 M. As expected, these values are similar to each other since the lanthanides are both of similar charge density and size. These data also indicate that the lanthanides are considerably more tightly bound than the biologically relevant metal Ca 2ϩ (binding constant for Ca 2ϩ ϭ 2 mM (19)) and that the presence of 1.5 mM Ca 2ϩ has very little effect on the overall dissociation constant of the trivalent lanthanides, most likely due to the preferential Coulombic interactions between the trivalent ions and the carboxylate-rich metal binding site.…”

Lanthanide-binding helix-turn-helix peptides: Solution structure of a designed metallonuclease

“…1). This peptide binds one equivalent of Ca(II) or lanthanide ions with binding affinities typical of EF-hand-derived peptides (26)(27)(28)(29)(30)(31).…”

Change in permeant size selectivity by phosphorylation of connexin 43 gap-junctional hemichannels by PKC

“…We measured LRET between Cx43 subunits labeled with either Tb 3ϩ or fluorescein to determine the composition of hemichannels based on the number of acceptor-labeled monomers per hemichannel. LRET is sensitive and independent of the connexin molecular weight and has been used in a few studies of membrane proteins to assess intersubunit and intramolecular distances (32)(33)(34)(35). LRET has many advantages for our experiments when compared with traditional FRET (31,32).…”