Nonfluorescent quenchers to correlate single-molecule conformational and compositional dynamics

Abstract

Single-molecule Förster resonance energy transfer (smFRET) is a powerful method for studying the conformational dynamics of a biomolecule in real-time. However, studying how interacting ligands correlate with and regulate the conformational dynamics of the biomolecule is extremely challenging because of the availability of a limited number of fluorescent dyes with both high quantum yield and minimal spectral overlap. Here we report the use of a nonfluorescent quencher (Black Hole Quencher, BHQ) as an acceptor for smFRET. Using a Cy3/BHQ pair, we can accurately follow conformational changes of the ribosome during elongation in real time. We demonstrate the application of single-color FRET to correlate the conformational dynamics of the ribosome with the compositional dynamics of tRNA. We use the normal Cy5 FRET acceptor to observe arrival of a fluorescently labeled tRNA with a concomitant transition of the ribosome from the locked to the unlocked conformation. Our results illustrate the potential of nonfluorescent quenchers in single-molecule correlation studies.

Figure 1

(a) Chemical structure of BHQ-2 conjugated to an oligonucleotide. The emission spectrum of Cy3 with absorption spectrum of BHQ-2 shows large spectral overlap for efficient energy transfer during FRET. (b) Intensity histogram of doubly-labeled oligonucleotides of varying inter-dye distance between Cy3 and BHQ. (c) Mean Cy3 fluorescence intensity as a function of inter-dye distance, along with the theoretical FRET curve. Error bars are 1 standard deviation. The mean intensity increases with increasing inter-dye distance, following the theoretical FRET curve within 1 standard deviation of error (resulting from the error in estimating I_MAX when calculating fluorescence intensity from FRET efficiency).

Figure 2

(a) Schematic of delivering BHQ-labeled 50S subunits with EF-G and ternary complex to pre-assembled PIC immobilized on PEG-covered quartz surface. (b) Representative time trace of fluorescence intensity for elongating ribosome translating mRNA 6(FK) (alternating Phe and Lys). On the right shows the histogram of the normalized number of elongation cycles observed for single ribosomes. (c). Representative time trace for elongating ribosome translating 6(FK) without Lys tRNA. Translation is stalled after the first codon. The small number of additional events beyond the first codon shown in the histogram is likely due to statistical errors in the identification of transitions by our analytical method.

Figure 3

(a) Comparison histogram of apparent translation efficiency at 80 nM TC (red) and 160 nM TC (blue). At higher [TC], the apparent translation efficiency is higher. (b) FRET state of the two conformational states, with a mean corresponding to 0.3 for the unlocked state and a mean of 0.49 for the locked state. (c) Mean lifetime (sec) of the locked state. The lifetime is lowered as [TC] is increased. (d) Mean lifetime (sec) of unlocked state. The lifetime is not dependent on [TC].

Figure 4

(a) Representative time trace of a correlation experiment, with the green trace (Cy3B) showing the ribosome locking and unlocking during elongation, and the red (Cy5) showing the arrival and departure of Phe-tRNA^Phe. (b) Post-synchronized time trace of the arrival of Phe-(Cy5)tRNA^Phe to the conformation change of the ribosome from locked to unlocked state. The arrival of tRNA is directly correlated with the unlocking of the ribosome. The unlocking of the ribosome at the second codon is not correlated with a Cy5 pulse, consistent with the mRNA sequence.