A photoprotection strategy for microsecond-resolution single-molecule fluorescence spectroscopy

Campos, Luis Alberto; Liu, Jianwei; Wang, X.; Ramanathan, Ravishankar; English, Douglas S.; Muñoz, Víctor

doi:10.1038/nmeth.1553

Cited by 112 publications

(134 citation statements)

References 18 publications

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“…We used A488-A594 as the SM-FRET pair. We have previously tested this same strategy on the slow two-state folder α-spectrin SH3 domain (55 residues), for which we could fully resolve the folded and unfolded states and found excellent quantitative agreement between bulk and SM-FRET experiments (32). In addition, we performed all the experiments at 279 K because at this temperature the BBL folding relaxation greatly slows down (21), which should facilitate reaching T b < τ f conditions.…”

Section: Resultsmentioning

confidence: 99%

“…In free-diffusing SM-FRET experiments, we managed to measure FEH from individual BBL molecules with 50-μs resolution across the entire chemical-denaturation curve thanks to a recently developed photoprotection cocktail (32). The results are shown in Fig.…”

Section: Nm (Assuming κmentioning

confidence: 99%

“…Current SM-FRET dyes saturate at photon emissions of approximately 100 kHz (31), restricting the effective time resolution to the 0.5-1 ms it takes to detect statistically significant numbers of photons. We optimized the time-resolution/ photodamage trade-off by performing chemical-denaturation experiments at 279 K (which slows down BBL kinetics by approximately 200-fold) in conjunction with a newly developed photoprotection cocktail that affords emissions of approximately one photon per microsecond with little photodamage (32). The other issue refers to the possible role of the protein tails in BBL folding, which we address by studying a variant of BBL labeled with single-molecule dyes at the ends of five-residue unstructured N-and C-terminal tails.…”

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confidence: 99%

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Exploring one-state downhill protein folding in single molecules

Liu

Campos

Cerminara

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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A one-state downhill protein folding process is barrierless at all conditions, resulting in gradual melting of native structure that permits resolving folding mechanisms step-by-step at atomic resolution. Experimental studies of one-state downhill folding have typically focused on the thermal denaturation of proteins that fold near the speed limit (ca. 10 6 s −1 ) at their unfolding temperature, thus being several orders of magnitude too fast for current single-molecule methods, such as single-molecule FRET. An important open question is whether one-state downhill folding kinetics can be slowed down to make them accessible to single-molecule approaches without turning the protein into a conventional activated folder. Here we address this question on the small helical protein BBL, a paradigm of one-state downhill thermal (un)folding. We decreased 200-fold the BBL folding-unfolding rate by combining chemical denaturation and low temperature, and carried out freediffusion single-molecule FRET experiments with 50-μs resolution and maximal photoprotection using a recently developed Troloxcysteamine cocktail. These experiments revealed a single conformational ensemble at all denaturing conditions. The chemical unfolding of BBL was then manifested by the gradual change of this unique ensemble, which shifts from high to low FRET efficiency and becomes broader at increasing denaturant. Furthermore, using detailed quantitative analysis, we could rule out the possibility that the BBL single-molecule data are produced by partly overlapping folded and unfolded peaks. Thus, our results demonstrate the onestate downhill folding regime at the single-molecule level and highlight that this folding scenario is not necessarily associated with ultrafast kinetics. P rotein folding is an ideal problem for single-molecule approaches because the simple collective behavior that is frequently observed in bulk experiments could hide an underlying complexity of myriads of microscopic folding pathways (1). Thus protein folding has been a major target for modern single-molecule experiments, including force-microscopy (2) and fluorescence (3). Among these, single-molecule FRET (SM-FRET) has the advantage of recapitulating the conventional bulk chemical unfolding experiments at the single-molecule level. SM-FRET methods have already made important contributions to protein folding, such as demonstrating the conversion between native and unfolded populations of two-state-like folding (4), resolving the chemical-denaturant-induced expansion of the unfolded state (5) and its nanosecond conformational dynamics (6), and setting upper bounds for folding transition-path times (7).Another important application is the characterization of the downhill folding scenario predicted by energy landscape theory (1). Downhill folding proteins have a maximal free-energy barrier (i.e., at the denaturation midpoint) below 3RT. (RT is thermal energy, where R is the gas constant and T is the temperature in Kelvin.) The barrier top is thus significantly populated, and ...

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Section: Resultsmentioning

confidence: 99%

Section: Nm (Assuming κmentioning

confidence: 99%

mentioning

confidence: 99%

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Exploring one-state downhill protein folding in single molecules

Liu

Campos

Cerminara

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…YOYO-1 was added to the imaging buffer and images were taken at a rate of 20 s Optimizing the buffer-conditions with respect to brightness and binding kinetics can be used to greatly increase the resolution. Several strategies have been reported to enhance the brightness and lifetime of fluorophores that rely on quenching of the triplet state or other dark states by chemical reactions [10][11][12][13][14] . In our case, both the addition of β-mercapto ethanol (BME) (Fig.…”

Section: Manuscript Textmentioning

confidence: 99%

Binding-Activated Localization Microscopy of DNA Structures

et al. 2011

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Many nucleic acid stains show a strong fluorescence enhancement upon binding to doublestranded DNA. Here we exploit this property to perform superresolution microscopy based on the localization of individual binding events. The dynamic labeling scheme and the optimization of fluorophore brightness yielded a resolution of 14 nm (fwhm) and a spatial sampling of 1/nm. We illustrate our approach with two different DNA-binding dyes and apply it to visualize the organization of the bacterial chromosome in fixed Escherichia coli cells. In general, the principle of binding-activated localization microscopy (BALM) can be extended to other dyes and targets such as protein structures.

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“…Benefitting from advances in general microscopy[1], detection devices [8], and fluorophore physics [9,10], single-molecule fluorescence spectroscopy has reached sub-nanometer spatial resolution [11] and microsecond temporal resolution [12,13]. Single-molecule fluorescence imaging is carried out primarily with total internal reflection, confocal, and zero-mode waveguide microscopy [2].…”

Section: Single-molecule Protein Studiesmentioning

confidence: 99%

Bringing single-molecule spectroscopy to macromolecular protein complexes

Joo

Fareh

Kim

2013

Trends in Biochemical Sciences

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Single-molecule fluorescence spectroscopy offers real-time, nanometer-resolution information. Over the past two decades, this emerging single-molecule technique has been rapidly adopted to investigate the structural dynamics and biological functions of proteins. Despite this remarkable achievement, single-molecule fluorescence techniques must be extended to macromolecular protein complexes that are physiologically more relevant for functional studies. In this review, we present recent major breakthroughs for investigating protein complexes within cell extracts using single-molecule fluorescence. We outline the challenges, future prospects and potential applications of these new single-molecule fluorescence techniques in biological and clinical research. Single-molecule protein studiesSingle-molecule techniques have become potent tools for the discovery of novel protein mechanisms by allowing high spatiotemporal resolution. Sub-nanometer resolution, the ultimate scale of biological systems, has been reached with single-molecule fluorescence microscopy[1] and spectroscopy [2]; single-molecule force and torque spectroscopy [3,4]; atomic force microscopy [5] and spectroscopy [6]; and nanopores [7]. Measurements can be carried out on the biologically relevant time scales of microseconds to minutes.Among these techniques, single-molecule fluorescence spectroscopy has enabled researchers to unveil the action mechanism of a protein by imaging protein activity in real time. Benefitting from advances in general microscopy[1], detection devices [8], and fluorophore physics [9,10], single-molecule fluorescence spectroscopy has reached sub-nanometer spatial resolution [11] and microsecond temporal resolution [12,13]. Single-molecule fluorescence imaging is carried out primarily with total internal reflection, confocal, and zero-mode waveguide microscopy [2]. Among these, total internal reflection fluorescence microscopy has been employed by several research teams in the development of novel techniques described in this review [14][15][16][17] Single-molecule observations using cell extractsUnlike purified recombinant proteins, crude cell extracts provide more biologically relevant environment in which molecules of interest can interact not only with their partners but also with other cellular components. It was anticipated to combine this approach with single molecule techniques and observe the assembly and function of macromolecular complexes in real time. However, technical hurdles related to the specificity and efficiency of labeling molecules of interest within crude cell extracts had been reported [22][23][24]. In order to overcome this limitation, several groups recently developed original approaches. Hoskins et al. used protein complexes specifically tagged with fluorophores [14,19], and Yardimci et al. immunostained reaction products using specific fluorescent antibodies [15,20]. Real-time observation of spliceosome assemblyDespite the relatively small number of genes in the human genome, we have extraordin...

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A photoprotection strategy for microsecond-resolution single-molecule fluorescence spectroscopy

Cited by 112 publications

References 18 publications

Exploring one-state downhill protein folding in single molecules

Exploring one-state downhill protein folding in single molecules

Binding-Activated Localization Microscopy of DNA Structures

Bringing single-molecule spectroscopy to macromolecular protein complexes

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