Buffer-Dependent Photophysics of 2-Aminopurine: Insights into Fluorescence Quenching and Excited-State Interactions.

2-Aminopurine (2AP) is the most widely used fluorescent nucleobase analogue in DNA and RNA research. Its unique photophysical properties and sensitivity to environmental changes make it a useful tool for understanding nucleic acid dynamics and DNA-protein interactions. We studied the effect of ions present in commonly used buffer solutions on the excited-state photophysical properties of 2AP. Fluorescence quenching was negligible for tris(hydroxymethyl)aminomethane (TRIS), but significant for phosphate, carbonate, 3-(N-morpholino) propanesulfonic acid (MOPS), and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffers. Results indicate that the two tautomers of 2AP (7H, 9H) are quenched by phosphate ions to different extents. Quenching by the H2PO4- ion is more pronounced for the 7H tautomer, while the opposite is true for the HPO42- ion. For phosphate ions, the results of the time-resolved fluorescence study cannot be explained using a simple collisional quenching mechanism. Instead, results are consistent with transient interactions between 2AP and the phosphate ions. We postulate that excited-state interactions between the 2AP tautomers and an H-bond acceptor (phosphate and carbonate) result in significant quenching of the singlet-excited state of 2AP. Such interactions manifest in biexponential fluorescence intensity decays with pre-exponential factors that vary with quencher concentration, and downward curvatures of the Stern-Volmer plots.

A new twist on PIFE: photoisomerisation-related fluorescence enhancement.

PIFE was first used as an acronym for protein-induced fluorescence enhancement, which refers to the increase in fluorescence observed upon the interaction of a fluorophore, such as a cyanine, with a protein. This fluorescence enhancement is due to changes in the rate ofcis/transphotoisomerisation. It is clear now that this mechanism is generally applicable to interactions with any biomolecule. In this review, we propose that PIFE is thereby renamed according to its fundamental working principle as photoisomerisation-related fluorescence enhancement, keeping the PIFE acronym intact. We discuss the photochemistry of cyanine fluorophores, the mechanism of PIFE, its advantages and limitations, and recent approaches to turning PIFE into a quantitative assay. We provide an overview of its current applications to different biomolecules and discuss potential future uses, including the study of protein-protein interactions, protein-ligand interactions and conformational changes in biomolecules.

A new twist on PIFE: photoisomerisation-related fluorescence enhancement.

PIFE was first used as an acronym for protein-induced fluorescence enhancement, which refers to the increase in fluorescence observed upon the interaction of a fluorophore, such as a cyanine, with a protein. This fluorescence enhancement is due to changes in the rate of cis/trans photoisomerisation. It is clear now that this mechanism is generally applicable to interactions with any biomolecule and, in this review, we propose that PIFE is thereby renamed according to its fundamental working principle as photoisomerisation-related fluorescence enhancement, keeping the PIFE acronym intact. We discuss the photochemistry of cyanine fluorophores, the mechanism of PIFE, its advantages and limitations, and recent approaches to turn PIFE into a quantitative assay. We provide an overview of its current applications to different biomolecules and discuss potential future uses, including the study of protein-protein interactions, protein-ligand interactions and conformational changes in biomolecules.

Uracil-DNA glycosylase efficiency is modulated by substrate rigidity.

Uracil DNA-glycosylase (UNG) is a DNA repair enzyme that removes the highly mutagenic uracil lesion from DNA using a base flipping mechanism. Although this enzyme has evolved to remove uracil from diverse sequence contexts, UNG excision efficiency depends on DNA sequence. To provide the molecular basis for rationalizing UNG substrate preferences, we used time-resolved fluorescence spectroscopy, NMR imino proton exchange measurements, and molecular dynamics simulations to measure UNG specificity constants (kcat/KM) and DNA flexibilities for DNA substrates containing central AUT, TUA, AUA, and TUT motifs. Our study shows that UNG efficiency is dictated by the intrinsic deformability around the lesion, establishes a direct relationship between substrate flexibility modes and UNG efficiency, and shows that bases immediately adjacent to the uracil are allosterically coupled and have the greatest impact on substrate flexibility and UNG activity. The finding that substrate flexibility controls UNG efficiency is likely significant for other repair enzymes and has major implications for the understanding of mutation hotspot genesis, molecular evolution, and base editing.

Potassium Glutamate and Glycine Betaine Induce Self-Assembly of the PCNA and ß-Sliding Clamps.

Sliding clamps are oligomeric ring-shaped proteins that increase the efficiency of DNA replication. The stability of the Escherichia coli ß-clamp, a homodimer, is particularly remarkable. The dissociation equilibrium constant of the ß-clamp is of the order of 10 pM in buffers of moderate ionic strength. Coulombic electrostatic interactions have been shown to contribute to this remarkable stability. Increasing NaCl concentration in the assay buffer results in decreased dimer stability and faster subunit dissociation kinetics in a way consistent with simple charge-screening models. Here, we examine non-Coulombic ionic effects on the oligomerization properties of sliding clamps. We determined relative diffusion coefficients of two sliding clamps using fluorescence correlation spectroscopy. Replacing NaCl by KGlu, the primary cytoplasmic salt in E. coli, results in a decrease of the diffusion coefficient of these proteins consistent with the formation of protein assemblies. The UV-vis spectrum of the ß-clamp labeled with tetramethylrhodamine shows the characteristic absorption band of dimers of rhodamine when KGlu is present in the buffer. This suggests that KGlu induces the formation of assemblies that involve two or more rings stacked face-to-face. Results can be quantitatively explained on the basis of unfavorable interactions between KGlu and the functional groups on the protein surface, which drive biomolecular processes that bury exposed surface. Similar results were obtained with the Saccharomyces cerevisiae PCNA sliding clamp, suggesting that KGlu effects are not specific to the ß-clamp. Clamp association is also promoted by glycine betaine, a zwitterionic compound that accumulates intracellularly when E. coli is exposed to high concentrations of extracellular solute. Possible biological implications are discussed.

Evidence that the TRPV1 S1-S4 membrane domain contributes to thermosensing.

Sensing and responding to temperature is crucial in biology. The TRPV1 ion channel is a well-studied heat-sensing receptor that is also activated by vanilloid compounds, including capsaicin. Despite significant interest, the molecular underpinnings of thermosensing have remained elusive. The TRPV1 S1-S4 membrane domain couples chemical ligand binding to the pore domain during channel gating. Here we show that the S1-S4 domain also significantly contributes to thermosensing and couples to heat-activated gating. Evaluation of the isolated human TRPV1 S1-S4 domain by solution NMR, far-UV CD, and intrinsic fluorescence shows that this domain undergoes a non-denaturing temperature-dependent transition with a high thermosensitivity. Further NMR characterization of the temperature-dependent conformational changes suggests the contribution of the S1-S4 domain to thermosensing shares features with known coupling mechanisms between this domain with ligand and pH activation. Taken together, this study shows that the TRPV1 S1-S4 domain contributes to TRPV1 temperature-dependent activation.

Impact of Cyanine Conformational Restraint in the Near-Infrared Range.

Appending conformationally restraining ring systems to the cyanine chromophore creates exceptionally bright fluorophores in the visible range. Here, we report the application of this strategy in the near-infrared range through the preparation of the first restrained heptamethine indocyanine. Time-resolved absorption spectroscopy and fluorescence correlation spectroscopy verify that, unlike the corresponding parent unrestrained variant, the restrained molecule is not subject to photoisomerization. Notably, however, the room-temperature emission efficiency and the fluorescence lifetime of the restrained cyanine are not extended relative to the parent cyanine, even in viscous solvents. Thus, in contrast to prior reports, the photoisomerization of heptamethine cyanines does not contribute significantly to the excited-state chemistry of these molecules. We also find that the fluorescence lifetime of the restrained heptamethine cyanine is temperature-insensitive and significantly extended at moderately elevated temperatures relative to the parent cyanine. Finally, computational studies have been used to evaluate the impact of the conformational restraint on atomic and orbital structure across the cyanine series. These studies clarify the role of photoisomerization in the heptamethine cyanine scaffold and demonstrate the dramatic effect of restraint on the temperature sensitivity of these dyes.

Tutorial: measurement of fluorescence spectra and determination of relative fluorescence quantum yields of transparent samples.

The measurement of fluorescence spectra and the determination of fluorescence quantum yields in transparent samples are conceptually simple tasks, but these procedures are subject to several pitfalls that can lead to significant errors. Available technical reports and protocols often assume that the reader possesses a solid theoretical background in spectroscopy and has ample experience with fluorescence instrumentation, but this is often not the case given the many applications of fluorescence in diverse fields of science. The goal of this tutorial is to provide a didactic treatment of the topic that will hopefully be accessible to readers without extensive expertise in the field of fluorescence. The article covers the theoretical background needed to understand the origins of the most common artifacts researchers can expect. Possible artifacts are illustrated with examples to help readers avoid them or identify them if present. A step-by-step example of a fluorescence quantum yield determination in solution is provided with detailed experimental information to help readers understand how to design and analyze experiments.

Pitching single-focus confocal data analysis one photon at a time with Bayesian nonparametrics.

Fluorescence time traces are used to report on dynamical properties of molecules. The basic unit of information in these traces is the arrival time of individual photons, which carry instantaneous information from the molecule, from which they are emitted, to the detector on timescales as fast as microseconds. Thus, it is theoretically possible to monitor molecular dynamics at such timescales from traces containing only a sufficient number of photon arrivals. In practice, however, traces are stochastic and in order to deduce dynamical information through traditional means-such as fluorescence correlation spectroscopy (FCS) and related techniques-they are collected and temporally autocorrelated over several minutes. So far, it has been impossible to analyze dynamical properties of molecules on timescales approaching data acquisition without collecting long traces under the strong assumption of stationarity of the process under observation or assumptions required for the analytic derivation of a correlation function. To avoid these assumptions, we would otherwise need to estimate the instantaneous number of molecules emitting photons and their positions within the confocal volume. As the number of molecules in a typical experiment is unknown, this problem demands that we abandon the conventional analysis paradigm. Here, we exploit Bayesian nonparametrics that allow us to obtain, in a principled fashion, estimates of the same quantities as FCS but from the direct analysis of traces of photon arrivals that are significantly smaller in size, or total duration, than those required by FCS.

Photophysical properties of the hemicyanine Dy-630 and its potential as a single-molecule fluorescent probe for biophysical applications.

Protein-induced fluorescence enhancement (PIFE) is an increasingly used approach to investigate DNA-protein interactions at the single molecule level. The optimal probe for this type of application is highly photostable, has a high absorption extinction coefficient, and has a moderate fluorescence quantum yield that increases significantly when the dye is in close proximity to a large macromolecule such as a protein. So far, the green-absorbing symmetric cyanine known as Cy3 has been the probe of choice in this field because the magnitude of the increase observed upon protein binding (usually 2-4 -fold) is large enough to allow for the analysis of protein dynamics on the inherently noisy single-molecule signals. Here, we report the characterization of the photophysical properties of the red-absorbing hemicyanine dye Dy-630 in the context of its potential application as a single-molecule PIFE probe. The behavior of Dy-630 in solution is similar to that of Cy3; the fluorescence quantum yield and lifetime of Dy-630 increase with increasing viscosity, and decrease with increasing temperature indicating the existence of an activated nonradiative process that depopulates the singlet state of the dye. As in the case of Cy3, the results of transient spectroscopy experiments are consistent with the formation of a photoisomer that reverts to the ground state thermally in the microsecond timescale. Unfortunately, experiments with DNA samples paint a more complex scenario. As in the case of Cy3, the fluorescence quantum yield of Dy-630 increases significantly when the dye interacts with the DNA bases, but in the case of Dy-630 attachment to DNA results in an already long fluorescence lifetime that does not provide a significant window for the protein-induced enhancement observed with Cy3. Although we show that Dy-630 may not be well-suited for PIFE, our results shed light on the optimal design principles for probes for PIFE applications.

An alternative framework for fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS), is a widely used tool routinely exploited for in vivo and in vitro applications. While FCS provides estimates of dynamical quantities, such as diffusion coefficients, it demands high signal to noise ratios and long time traces, typically in the minute range. In principle, the same information can be extracted from microseconds to seconds long time traces; however, an appropriate analysis method is missing. To overcome these limitations, we adapt novel tools inspired by Bayesian non-parametrics, which starts from the direct analysis of the observed photon counts. With this approach, we are able to analyze time traces, which are too short to be analyzed by existing methods, including FCS. Our new analysis extends the capability of single molecule fluorescence confocal microscopy approaches to probe processes several orders of magnitude faster and permits a reduction of photo-toxic effects on living samples induced by long periods of light exposure.

Protein Environment and DNA Orientation Affect Protein-Induced Cy3 Fluorescence Enhancement.

The cyanine dye Cy3 is a popular fluorophore used to probe the binding of proteins to nucleic acids as well as their conformational transitions. Nucleic acids labeled only with Cy3 can often be used to monitor interactions with unlabeled proteins because of an enhancement of Cy3 fluorescence intensity that results when the protein contacts Cy3, a property sometimes referred to as protein-induced fluorescence enhancement (PIFE). Although Cy3 fluorescence is enhanced upon contacting most proteins, we show here in studies of human replication protein A and Escherichia coli single-stranded DNA binding protein that the magnitude of the Cy3 enhancement is dependent on both the protein as well as the orientation of the protein with respect to the Cy3 label on the DNA. This difference in PIFE is due entirely to differences in the final protein-DNA complex. We also show that the origin of PIFE is the longer fluorescence lifetime induced by the local protein environment. These results indicate that PIFE is not a through space distance-dependent phenomenon but requires a direct interaction of Cy3 with the protein, and the magnitude of the effect is influenced by the region of the protein contacting Cy3. Hence, use of the Cy3 PIFE effect for quantitative studies may require careful calibration.

Publisher Correction: Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study.

This paper was originally published under standard Springer Nature copyright. As of the date of this correction, the Analysis is available online as an open-access paper with a CC-BY license. No other part of the paper has been changed.

Assembly-disassembly is coupled to the ATPase cycle of tobacco Rubisco activase.

The carbon-fixing activity of enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is regulated by Rubisco activase (Rca), a ring-forming ATPase that catalyzes inhibitor release. For higher plant Rca, the catalytic roles played by different oligomeric species have remained obscure. Here, we utilized fluorescence-correlation spectroscopy to estimate dissociation constants for the dimer-tetramer, tetramer-hexamer, hexamer-12-mer, and higher-order assembly equilibria of tobacco Rca. A comparison of oligomer composition with ATPase activity provided evidence that assemblies larger than hexamers are hydrolytically inactive. Therefore, supramolecular aggregates may serve as storage forms at low-energy charge. We observed that the tetramer accumulates only when both substrate and product nucleotides are bound. During rapid ATP turnover, about one in six active sites was occupied by ADP, and ∼36% of Rca was tetrameric. The steady-state catalytic rate reached a maximum between 0.5 and 2.5 µm Rca. In this range, significant amounts of dimers, tetramers, and hexamers coexisted, although none could fully account for the observed activity profile. Therefore, we propose that dynamic assembly-disassembly partakes in the ATPase cycle. According to this model, the association of dimers with tetramers generates a hexamer that forms a closed ring at high ATP and magnesium levels. Upon hydrolysis and product release, the toroid breaks open and dissociates into a dimer and tetramer, which may be coupled to Rubisco remodeling. Although a variant bearing the R294V substitution assembled in much the same way, highly stabilized states could be generated by binding of a transition-state analog. A tight-binding pre-hydrolysis state appears to become more accessible in thermally labile Rcas.

Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study.

Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods.

Photophysical characterization of interchromophoric interactions between rhodamine dyes conjugated to proteins.

Rhodamine dyes in aqueous solution form non-fluorescent dimers with a plane-to-plane stacking geometry (H-dimers). The self-quenching properties of these dimers have been exploited to probe the conformation and dynamics of proteins using a variety of fluorescence approaches that require the interpretation of fluorescence intensities, lifetimes and fluctuations. Here, we report on a systematic study of the photophysical properties of three rhodamine dyes (tetramethylrhodamine, Alexa 488 and Alexa 546) covalently bound to the E. coli sliding clamp (ß clamp) with emphasis on the properties of the H-dimers that form when the dimeric protein is labeled with one dye at each side of the dimer interface. Overall, results are consistent with an equilibrium between non-emissive dimers and unstacked monomers that experience efficient dynamic quenching Protein constructs labeled with tetramethylrhodamine show the characteristic features of H-dimers in their absorption spectra and a c.a. 40-fold quenching of fluorescence intensity. The degree of quenching decreases when samples are labeled with a tetramethylrhodamine derivative bearing a six-carbon linker. H-dimers do not form in samples labeled with Alexa 488 and A546, but fluorescence is still quenched in these samples through a dynamic mechanism. These results should help researchers design and interpret fluorescence experiments that take advantage of the properties of rhodamine dimers in protein research.