Taekjip Ha

A Practical Guide to Single Molecule FRET

Single-molecule fluorescence resonance energy transfer (smFRET) is one of the most general and adaptable single-molecule techniques. Despite the explosive growth in the application of smFRET to answer biological questions in the last decade, the technique has been practiced mostly by biophysicists. We provide a practical guide to using smFRET, focusing on the study of immobilized molecules that allow measurements of single-molecule reaction trajectories from 1 ms to many minutes. We discuss issues a biologist must consider to conduct successful smFRET experiments, including experimental design, sample preparation, single-molecule detection and data analysis. We also describe how a smFRET-capable instrument can be built at a reasonable cost with off-the-shelf components and operated reliably using well-established protocols and freely available software.

Measuring mechanical tension across vinculin reveals regulation of focal adhesion dynamics

Mechanical forces are central to developmental, physiological and pathological processes. However, limited understanding of force transmission within sub-cellular structures is a major obstacle to unravelling molecular mechanisms. Here we describe the development of a calibrated biosensor that measures forces across specific proteins in cells with piconewton (pN) sensitivity, as demonstrated by single molecule fluorescence force spectroscopy. The method is applied to vinculin, a protein that connects integrins to actin filaments and whose recruitment to focal adhesions (FAs) is force-dependent. We show that tension across vinculin in stable FAs is ∼2.5 pN and that vinculin recruitment to FAs and force transmission across vinculin are regulated separately. Highest tension across vinculin is associated with adhesion assembly and enlargement. Conversely, vinculin is under low force in disassembling or sliding FAs at the trailing edge of migrating cells. Furthermore, vinculin is required for stabilizing adhesions under force. Together, these data reveal that FA stabilization under force requires both vinculin recruitment and force transmission, and that, surprisingly, these processes can be controlled independently.

Advances in Single-Molecule Fluorescence Methods for Molecular Biology

Ever since their introduction two decades ago, single-molecule (SM) fluorescence methods have matured and branched out to address numerous biological questions, which were inaccessible via ensemble measurements. Among the current arsenal, SM fluorescence techniques have capabilities of probing the dynamic interactions of nucleic acids and proteins via Förster (fluorescence) resonance energy transfer (FRET), tracking single particles over microns of distances, and deciphering the rotational motion of multisubunit systems. In this exciting era of transitioning from in vitro to in vivo and in situ conditions, it is anticipated that SM fluorescence methodology will become a common tool of molecular biology.

Nonblinking and long-lasting single-molecule fluorescence imaging

Photobleaching and blinking of fluorophores pose fundamental limitations on the information content of single-molecule fluorescence measurements. Photoinduced blinking of Cy5 has hampered many previous investigations using this popular fluorophore. Here we show that Trolox in combination with the enzymatic oxygen-scavenging system eliminates Cy5 blinking, dramatically reduces photobleaching and improves the signal linearity at high excitation rates, significantly extending the applicability of single-molecule fluorescence techniques.

Probing Single-Stranded DNA Conformational Flexibility Using Fluorescence Spectroscopy

Single-stranded DNA (ssDNA) is an essential intermediate in various DNA metabolic processes and interacts with a large number of proteins. Due to its flexibility, the conformations of ssDNA in solution can only be described using statistical approaches, such as flexibly jointed or worm-like chain models. However, there is limited data available to assess such models quantitatively, especially for describing the flexibility of short ssDNA and RNA. To address this issue, we performed FRET studies of a series of oligodeoxythymidylates, (dT)N, over a wide range of salt concentrations and chain lengths (10 < or = N < or = 70 nucleotides), which provide systematic constraints for testing theoretical models. Unlike in mechanical studies where available ssDNA conformations are averaged out during the time it takes to perform measurements, fluorescence lifetimes may act here as an internal clock that influences fluorescence signals depending on how fast the ssDNA conformations fluctuate. A reasonably good agreement could be obtained between our data and the worm-like chain model provided that limited relaxations of the ssDNA conformations occur within the fluorescence lifetime of the donor. The persistence length thus estimated ranges from 1.5 nm in 2 M NaCl to 3 nm in 25 mM NaCl.

Initiation and re-initiation of DNA unwinding by the Escherichia coli Rep helicase

Helicases are motor proteins that couple conformational changes induced by ATP binding and hydrolysis with unwinding of duplex nucleic acid, and are involved in several human diseases. Some function as hexameric rings, but the functional form of non-hexameric helicases has been debated. Here we use a combination of a surface immobilization scheme and single-molecule fluorescence assays—which do not interfere with biological activity—to probe DNA unwinding by the Escherichia coli Rep helicase. Our studies indicate that a Rep monomer uses ATP hydrolysis to move toward the junction between single-stranded and double-stranded DNA but then displays conformational fluctuations that do not lead to DNA unwinding. DNA unwinding initiates only if a functional helicase is formed via additional protein binding. Partial dissociation of the functional complex during unwinding results in interruptions (‘stalls’) that lead either to duplex rewinding upon complete dissociation of the complex, or to re-initiation of unwinding upon re-formation of the functional helicase. These results suggest that the low unwinding processivity observed in vitro for Rep is due to the relative instability of the functional complex. We expect that these techniques will be useful for dynamic studies of other helicases and protein–DNA interactions.

Spontaneous intersubunit rotation in single ribosomes.

During the elongation cycle, tRNA and mRNA undergo coupled translocation through the ribosome catalyzed by elongation factor G (EF-G). Cryo-EM reconstructions of certain EF-G-containing complexes led to the proposal that the mechanism of translocation involves rotational movement between the two ribosomal subunits. Here, using single-molecule FRET, we observe that pretranslocation ribosomes undergo spontaneous intersubunit rotational movement in the absence of EF-G, fluctuating between two conformations corresponding to the classical and hybrid states of the translocational cycle. In contrast, posttranslocation ribosomes are fixed predominantly in the classical, nonrotated state. Movement of the acceptor stem of deacylated tRNA into the 50S E site and EF-G binding to the ribosome both contribute to stabilization of the rotated, hybrid state. Furthermore, the acylation state of P site tRNA has a dramatic effect on the frequency of intersubunit rotation. Our results provide direct evidence that the intersubunit rotation that underlies ribosomal translocation is thermally driven.

Cytosolic viral sensor RIG-I is a 5'-triphosphate-dependent translocase on double-stranded RNA.

Retinoic acid inducible–gene I (RIG-I) is a cytosolic multidomain protein that detects viral RNA and elicits an antiviral immune response. Two N-terminal caspase activation and recruitment domains (CARDs) transmit the signal, and the regulatory domain prevents signaling in the absence of viral RNA. 5′-triphosphate and double-stranded RNA (dsRNA) are two molecular patterns that enable RIG-I to discriminate pathogenic from self-RNA. However, the function of the DExH box helicase domain that is also required for activity is less clear. Using single-molecule protein-induced fluorescence enhancement, we discovered a robust adenosine 5′-triphosphate–powered dsRNA translocation activity of RIG-I. The CARDs dramatically suppress translocation in the absence of 5′-triphosphate, and the activation by 5′-triphosphate triggers RIG-I to translocate preferentially on dsRNA in cis. This functional integration of two RNA molecular patterns may provide a means to specifically sense and counteract replicating viruses.

Probing cellular protein complexes using single-molecule pull-down

Proteins perform most cellular functions in macromolecular complexes. The same protein often participates in different complexes to exhibit diverse functionality. Current ensemble approaches of identifying cellular protein interactions cannot reveal physiological permutations of these interactions. Here we describe a single-molecule pull-down (SiMPull) assay that combines the principles of a conventional pull-down assay with single-molecule fluorescence microscopy and enables direct visualization of individual cellular protein complexes. SiMPull can reveal how many proteins and of which kinds are present in the in vivo complex, as we show using protein kinase A. We then demonstrate a wide applicability to various signalling proteins found in the cytosol, membrane and cellular organelles, and to endogenous protein complexes from animal tissue extracts. The pulled-down proteins are functional and are used, without further processing, for single-molecule biochemical studies. SiMPull should provide a rapid, sensitive and robust platform for analysing protein assemblies in biological pathways.

Orientation dependence in fluorescent energy transfer between Cy3 and Cy5 terminally attached to double-stranded nucleic acids

We have found that the efficiency of fluorescence resonance energy transfer between Cy3 and Cy5 terminally attached to the 5′ ends of a DNA duplex is significantly affected by the relative orientation of the two fluorophores. The cyanine fluorophores are predominantly stacked on the ends of the helix in the manner of an additional base pair, and thus their relative orientation depends on the length of the helix. Observed fluorescence resonance energy transfer (FRET) efficiency depends on the length of the helix, as well as its helical periodicity. By changing the helical geometry from B form double-stranded DNA to A form hybrid RNA/DNA, a marked phase shift occurs in the modulation of FRET efficiency with helix length. Both curves are well explained by the standard geometry of B and A form helices. The observed modulation for both polymers is less than that calculated for a fully rigid attachment of the fluorophores. However, a model involving lateral mobility of the fluorophores on the ends of the helix explains the observed experimental data. This has been further modified to take account of a minor fraction of unstacked fluorophore observed by fluorescent lifetime measurements. Our data unequivocally establish that Förster transfer obeys the orientation dependence as expected for a dipole–dipole interaction.