Resonance Energy Transfer Between Luminescent Quantum Dots and Diverse Fluorescent Protein Acceptors

Abstract

We characterized the resonance energy transfer interactions for conjugates consisting of QD donors self-assembled with three distinct fluorescent protein acceptors: two monomeric fluorescent proteins, the dsRed derivative mCherry or yellow fluorescent protein and the multi-chromophore b-phycoerythrin light harvesting complex. Using steady-state and time-resolved fluorescence, we showed that nonradiative transfer of excitation energy in these conjugates can be described within the Förster dipole-dipole formalism, with transfer efficiencies that vary with the degree of spectral overlap, donor-acceptor separation distance and the number of acceptors per QD. Comparison between the quenching data and simulation of the conjugate structures indicated that while energy transfer to monomeric proteins was identical to what was measured for QD-dye pairs, interactions with b-phycoerythrin were more complex. For the latter, the overall transfer efficiency results from the cumulative contribution of individual channels between the central QD and the chromophores distributed throughout the protein structure. Due to the biocompatible nature of fluorescent proteins, these QD-assemblies may have great potential for use in intracellular imaging and sensing.

Figure 1. Photophysical characteristics of the fluorophores

Normalized absorption and PL spectra of the 510 nm QD-YFP pair (A), 550 nm QD-mCherry pair (B), and 520 nm QD-b-PE and 540 nm QD-b-PE pairs (C). Spectral overlap functions for the four pairs are shown in panel (D). The inset shows a close up of the QD-YFP and QD-mCherry overlap functions.

Figure 2. Fluorescence spectra and quenching efficiencies

(A-C) Representative composite and deconvoluted spectra showing individual PL contributions as a function of the protein-to-QD ratio for: 510 nm QD-YFP, 550 nm QD-mCherry, and 540 nm-QD-b-PE assemblies. (D-E) Plots of the corresponding quenching efficiencies along with the acceptor-sensitized emission. In panel (E), the energy transfer efficiency extracted from changes in the QD excited state lifetime are also shown for the 550 nm QD-mCherry pair. Data are corrected for the direct excitation of the b-PE. QD emission profiles were fit with a Gaussian function.

Figure 3. Time-resolved fluorescence data for 550 nm QD-mCherry conjugates

(A) Plot of 550 nm QD PL intensity (monitored at 537 nm) versus time for several mCherry-to-QD ratios. (B) Plot of mCherry intensity (monitored at 620 nm) versus time for several mCherry-to-QD ratios. (C) Superimposed plots of normalized QD and mCherry intensity vs. time for selected ratios.

Figure 4. Model structures for QD-YFP and QD-mCherry conjugates

(A) 510 nm QD-YFP structure. QD with radius of ~28 Å (blue) is surrounded by a DHLA shell of 10 Å (crimson). The YFP is attached to the QD via a double His₆ sequence; only the N-terminal His₆ is shown in green. The YFP residue attached to the double (His)₆ linker is shown in red. The central YFP chromophore is shown in magenta. The yellow line corresponds to the 62 Å experimental separation distance. (B) Rendition of a 550 nm QD-mCherry structure using 56 Å experimental separation distance (yellow), with the protein chromophore shown in red. The mCherry residue attached to the (His)₆-linker sequence is shown in green, but the 35 amino acid linker and terminal His₆ sequence are not shown.

Figure 5. Models of the QD-b-PE conjugate structure/conformation

(A) b-PE is parallel to the QD surface (θ_QD:b-PE = 0°), and (B) b-PE fully extended away from the QD (θ_QD:b-PE = 90°). Central QD with radius of ~29 Å (r_QD) shown in blue is surrounded by a crimson shell of ~25 Å thickness representing the DHLA-PEG-biotin. The intermediary Streptavidin (SA) protein is shown in yellow with biotin-binding sites highlighted in purple. The b-PE ring structure is shown in white with the multiple chromophores highlighted in red. The inner concentric white circle corresponds to the R₀ (53 Å) for the 540 nm-QD-b-PE assembly. The 2^nd outer white circle is a visual distance marker set at ~95 Å from the QD center and represents the closest approach of the b-PE to the QD.

Figure 6. Correlations between b-PE structure and FRET data

(A) Schematic representation of possible conformations. θ_QD:b-PE is the angle between the QD-b-PE center-to-center axis and the axis perpendicular to the b-PE structure. R_QD:b-PE is the QD-b-PE center to center separation distance. (B) QD-b-PE center-to-center distances estimated from FRET efficiencies and plotted versus R_QD:b-PE (correspond to 0° ≤ θ_QD:b-PE ≤ 90°) for 540 nm (circles) and 520 nm (triangles) QDs. (C) Schematic mapping of the FRET efficiency for the QD-b-PE conjugate showing the effects of distance and distribution of the chromophores in the protein. Two representative b-PE conformations (dotted) along with the region explored by all possible conformations sampled by the protein on the nanocrystal (dashed). The color intensities indicate the FRET efficiencies between the QD and individual dyes in the b-PE (see bar on the right).