Single-Molecule FRET Imaging: History
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Subjects: Virology
Contributor:
Maolin Lu

Single-molecule Förster resonance energy transfer (smFRET) has provided a powerful platform to connect structure–function in motion, revealing dynamic aspects of spikes for several viruses: SARS-CoV-2, HIV-1, influenza, and Ebola.

  • single-molecule imaging
  • Förster resonance energy transfer (FRET)
  • virus spike-host interactions
  • SARS-CoV-2
  • HIV-1
  • Dynamics
  • virus spikes

1. Introduction

Virus spikes on the surface of enveloped viruses are often also viral fusion proteins that mediate the fusion between viral membranes and cellular membranes (Figure 1) essential for virus entry [1][2][3]. The merging of virus and lipid bilayers progresses through a hemifusion intermediate, followed by a fusion pore widening, content mixing, and the delivery of virus capsids into the host cytoplasm [4]. Viral fusion proteins respond to the binding of cellular receptors or acidic pH to undergo conformational rearrangements, which eventually promote membrane fusion. Viral fusion proteins have been categorized into three classes [1][2], of which Class I viral fusion proteins include the medically important SARS-CoV-2 spike (S) protein, the HIV-1 envelope (Env) protein, influenza hemagglutinin (HA), and Ebola glycoprotein (GP). These virus spikes are first synthesized as trimers of a single-chain polypeptide—an immature precursor, then go through proteolytical processing by host proteases to form mature spikes—trimers of heterodimers (Figure 1A). Mature spikes are highly metastable on the virus surface. Upon interacting with hosts, mature spikes undergo conformational changes from pre-fusion conformations to the lowest-energy post-fusion conformation (a common hairpin-like or the analogous coiled-coil conformation) through hypothetical intermediates in which the fusion peptide extends and inserts into the host target membrane (Figure 1B). Numerous pre-fusion and post-fusion structures of virus spikes have provided unprecedented details of conformations at individual steps during the viral membrane fusion [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21]. The recent dynamic studies on virus spikes established platforms to connect these structural snapshots in real time, revealed the order and the kinetics of transitional events, and guided developing interventions aiming to arrest or block viral membrane fusion, thus stopping viral infection [22][23][24][25][26][27][28][29][30][31].

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Figure 1. Class I viral fusion proteins and proposed model of viral membrane fusion. (A) Schematic drawing of spike precursor and cleaved spike. The spike protein is initially synthesized as a single-chain polypeptide (spike precursor) and later cleaved into a trimer of covalently or non-covalently linked heterodimer. The heterodimer consists of the surface receptor-binding subunit (gray) and the fusion subunit (fusion peptide or fusion loop (FP/FL), dark yellow; N-terminal domain, cyan; C-terminal domain, dark blue). (B) Proposed conformational events of virus spikes during viral membrane fusion. These events are as follows, involving conformational changes in the surface subunit (top row) and changes in the fusion subunit (low row, simplified by only showing the fusion subunit [1]). (1) Prefusion—conformations of the spike in “closed” and open forms. Spike activation proceeds through an opening of the trimer, usually in response to binding to receptor or due to a cellular cue such as low pH. For non-covalently linked spikes, dissociating/decoupling between the surface/exterior subunit with the fusion subunit has been observed/suggested after the spike opens, such as HIV-1 and SARS-CoV-2 spikes. FP/FL remains sequestered in this process. (2) Exposing, extending, and inserting the FP/FL into the cellular membrane leads to the formation of an extended prehairpin intermediate. (3) Folding back the C-terminal segment of the fusion subunit back on the N-terminal segment core brings viral and cellular membranes into proximity. (4) Further folding and dragging two membranes into contact promotes two membranes’ merging to form a hemifusion stalk. (5) The fusion subunit folds into a stable post-fusion conformation, allowing a fusion pore to form. The intermediate steps from (2) to (4) remain elusive. This proposed model does not specify or speculate the number of spikes required for fusion pore formation.

As spikes are highly exposed to our immune system, they are main targets of neutralizing antibodies and thus are critical for developing vaccines and anti-spike therapeutics. Most vaccines or vaccine candidates for HIV-1/AIDS and SARS-CoV-2/COVID-19 are based on their spike proteins to trigger the immune system to produce neutralizing antibodies. Interestingly, in the face of immune pressure, many viral spike proteins use conformational masking of vulnerable antibody-targeted epitopes. In addition to glycan shields and hypermutations, this strategy of conformational masking has been best-understood in HIV-1 [32][33][34][35][36][37].

Collectively, virus spike proteins are structurally flexible and conformationally dynamic on the virus surface. The dynamic nature and conformational plasticity of spikes enables entry of enveloped viruses into host target cells through membrane fusion, while distracting the immune system from antibody recognition. Molecular understanding and modulation of spikes’ intrinsic dynamics can guide the rational design of spike-targeting interventions such as vaccines, small-molecule inhibitors, and antibodies to block virus entry. Single-molecule Förster resonance energy transfer (smFRET), a spectroscopic tool sensitive to inter-fluorophore molecular distances, has been well-positioned to capture the innate dynamics of virus spikes labeled with Förster resonance energy transfer (FRET)-paired fluorophores. This minireview covers recent work on single-molecule FRET imaging of class I fusion proteins of enveloped viruses, including SARS-CoV-2 [31], HIV-1 [22][23][24][25][26][27], influenza [29], and Ebola [28][30].

2. Single-Molecule Förster Resonance Energy Transfer (smFRET) Imaging

Imaging macromolecules at the single-molecule/single-particle level has advanced our understanding of both static and dynamic aspects in virus–host interactions, merited by avoiding the averaging-out effect from traditional ensemble-level measurements. Those imaging techniques, such as single-particle cryoEM/cryoET, single-particle optical tracking, and super-resolution fluorescence microscopy exerted significant roles in addressing fundamental questions with regards to structures, dynamics, and functions of virus molecules underlying virus–host interactions [38][39][40][41][42][43][44].

Single-molecule Förster resonance energy transfer (smFRET) imaging has been proven to be a reliable imaging tool for revealing the intrinsically dynamic, heterogeneous nature of biological systems [45][46][47]. It is powerful to probe protein conformational dynamics in a time-resolved manner; identify previously unknown states; detect intermediates transient in nature; and delineate the sequence, the order, the timing, the frequency of conformational states, and the transitional events. Two significant advantages of smFRET are (1) it permits direct in situ observations of different conformations/structures of biological molecules in real time, and (2) it reveals conformational intermediates and features that are previously concealed or averaged-out in ensembles.

FRET refers to non-radiative energy transfer between a donor and acceptor fluorophore. The energy transfer efficiency is a function of distances between both fluorophores, described as FRET = 1/(1 + (R/R0)6), where R is the inter-fluorophore distance and R0 is the Förster distance determining the range of sensitive measure of distance [47]. For mostly used paired-fluorophores, the FRET-detectable distance ranges from 30 Å to 80 Å, well suited for the dimensions of spike proteins of enveloped viruses (Figure 2A). In application, a pair of donor and acceptor fluorophores is site-specifically labeled on the one or two molecules of interest. The donor fluorophores are excited by a laser, and the single-molecule fluorescence from both fluorophores is separately recorded for seconds to minutes by total internal fluorescence microscope (TIRF) or confocal microscopy [47], in which prism-based TIRF and objective TIRF are widely used (Figure 2B). Fluorescence and the quantified FRET values monitor the proximity between two fluorophores in real-time, ultimately translating to the object’s intra-molecular or inter-molecular dynamics (Figure 2C,D). The object of interest can be genetic materials, proteins, peptides, and other biomolecules. The applications of FRET in biological systems are broad, thanks to advances in instrumentations, analysis software, and site-specific dye-labeling methods. For instance, newly developed scientific CMOS (sCMOS) cameras and the developed smFRET software or algorisms [48][49][50][51] facilitate high-throughput smFRET imaging and robust data analysis.

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Figure 2. Single-molecule Förster resonance energy transfer (smFRET) principle, instrumentation, and imaging of dynamic biomolecules. (A) An example curve depicting energy transfer efficiency (Förster resonance energy transfer (FRET), dashed black line) from an excited donor fluorophore to a neighboring acceptor fluorophore as a function of donor–acceptor distances. FRET values or FRET negatively correlate with the distance within a couple of nanometers between a donor (yellow star) and an acceptor (red star). (B) Widely used smFRET imaging instrumentations: prism- and objective-based total internal reflection fluorescence (TIRF). (C,D) Real-time observations of conformational motions in biomolecules by smFRET. (C) Diagram depicting ideal FRET-derived space-time coordinates of biomolecule conformations. Donor, yellow star; acceptor, red star; relative fluorescence intensity, the star’s size; biomolecule of interest, gray. (D) Example FRET-related traces showing four interconvertible conformations in real time. The host molecule dynamically samples four conformations, reflected by different donor–acceptor energy transfer efficiencies. Donor fluorescence trace, solid yellow line; acceptor fluorescence trace, solid red line; calculated FRET trace, solid blue line; FRET-indicated conformations, dashed black lines.

In contrast, the attachment of fluorophores to specific sites on proteins without disturbing their functionality has been a technical obstacle. The conventional way to label proteins is to introduce cysteines, which permit maleimide-functionalized dyes’ attachments [52]. A considerable number of pre-existing essential cysteines on virus proteins, especially spike proteins, make it infeasible. Two alternative fluorophore-attaching strategies, enzymatic and amber-click labeling, have overcome this hurdle. Enzymatic methods take advantage of enzymes that site-specifically recognize short-peptide tags (6–12 amino acids in length) introduced into the protein of interest and transfer dye-conjugated substrates to these tags [53][54]. The genetically encoded copper-free click chemistry (amber-click) allows reading through introduced amber stop codons on the protein of interest as unnatural amino acids through amber suppression, followed by site-specifically labeling conjugated dyes on unnatural amino acids by copper-free click chemistry [55][56]. Both enzymatic and amber-click methods have been applied to study virus spikes for many enveloped viruses to reveal dynamic aspects during viral membrane fusion [22][23][24][25][26][27][28][29][30][31].

This entry is adapted from the peer-reviewed paper 10.3390/v13020332

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