Multicolor single-molecule FRET to explore protein folding and binding

Abstract

Proper protein function in cells, tissues and organisms depends critically on correct protein folding or interaction with partners. Over the last decade, single-molecule FRET (smFRET) has emerged as a powerful tool to probe complex distributions, dynamics, pathways and landscapes in protein folding and binding reactions, leveraging its ability to avoid averaging over an ensemble of molecules. While smFRET was practiced in a two-color form until recently, the last few years have seen the development of enhanced multicolor smFRET methods that provide additional structural information permitting us to probe more complex mechanisms. In this review, we provide a brief introduction to the smFRET technique, then follow with advanced multicolor measurements and end with ongoing methodology developments in microfluidics and protein labeling that are beginning to make these techniques more broadly applicable to answering a number of key questions about folding and binding.

Figure 1

Schematic depicting complex networks that comprise the folding and interactions of proteins. For example, the transition from unfolded to folded states can proceed directly or through intermediates, which can be partially folded or misfolded. Misfolding in proteins has been linked with aggregation-related diseases, where proteins self-assemble into toxic oligomers or fibrils. On the other hand, interactions with cellular partners can be crucial for protein activity; we depict here two possible mechanisms of coupled binding and folding, with structure formation either preceding binding or vice versa.

Figure 2

Multicolor smFRET study of the Rop mutants (adapted from reference 51: Gambin et al., Proc. Natl. Acad. Sci., USA, 2009, 106: 10153–10158) (A) The Repressor of Primers (Rop) complexes are formed by association of two helix-turn-helix motifs. The active enzyme requires that the two monomers bind in an anti conformation to form the proper RNA interface. (B) The symmetry created by multiple mutations within the hydrophobic core of the protein pushes the two monomers to re-arrange into a stable syn conformation, as observed for the A₂I₂ mutant. Surprisingly, conditions were found where the A₂L₂ mutant can form coexisting anti and syn, as evidenced by the presence of two FRET peaks. (C) Multi-color smFRET was used to check whether monomers are separating during the interconversion between the two opposite folded states. In a dual-channel format, the experiments took advantage of the difference between two possible dye pairs to detect in a simple manner the formation of mixed species in a competition assay (see text for details). The bottom left panel shows the FRET efficiencies of the syn and anti conformations for the Rop dimers forming an A488-A647 pair (green and red dyes) or forming an A488-A594 pair (green and purple dyes). The formation of A488-A594 pairs would only occur if the original A488-A647 dimers dissociate during the structural rearrangement. When we performed this test, we observed the two FRET peaks corresponding to the syn and anti conformations of A488-A647 dimers, and no significant higher-FRET species that would indicate exchange with A594 monomers (as found in a separate control experiment, bottom right).

Figure 3

Multicolor single-molecule FRET and coincidence (A) The structures of the Trp repressor dimer is used to illustrate the different pathways followed during protein-protein interaction and folding. Starting from two monomers kept in unfolded states (using for example denaturing conditions), one could trigger folding and observe in real time the steps of dimer formation. Two simple cases can be imagined: the monomers could fold separately then bind once the correct interface is formed, or first interact and together perform the folding steps. By using 3 fluorescent dyes, it becomes possible to distinguish between the two simple pathways, or even detect new intermediates. The first monomer would be labeled with the FRET pair in order to detect conformational changes; the second monomer would be labeled with a single dye, to detect simply its presence and the formation of a complex. (B) Schematic of single-molecule fluorescent bursts in the 3-color format. The highlighted bursts with high/low FRET and coincidence/no coincidence correspond to various folding/binding steps. In a 2D representation of FRET versus Coincidence, these different populations would be well separated and the sequence of folding/binding steps clearly identified. (C) 3-color smFRET histograms from proof-of-principle experiments with DNA (adapted from reference 62: Clamme et al., Chemphyschem, 2005, 6: 74–77).

Figure 4

Microfluidic platform for enhanced single-molecule FRET detection (adapted from reference : Lemke et al., J. Amer. Chem. Soc., 2009, 131: 13610–13612). (A) A laminar-flow mixer can remove denaturant or add binding partner to trigger folding events. (B) Large channels ventilated by nitrogen flank the channels containing buffers and proteins, efficiently removing oxygen from the solution before mixing. (C) smFRET histograms obtained with the device, showing (i) the folding of T4 lysozyme proteins upon removal of guanidinium chloride, (ii) the radical improvement of data upon deoxygenation for data from a near IR-dye (Alexa750) and (iii) for T4 lysozyme proteins labeled by using an unnatural amino-acid strategy.