FRET Dynamics Research Articles

Chemical friction along the minor groove of DNA facilitates enzymatic translocation of λ-exonuclease via electrostatic ratchet.

DNA is a long polymeric substrate that provides specific binding sites for many proteins in cell nucleus. Both diffusion and directed translocation play an essential role for protein-DNA interactions in gene regulation processes but it is still not fully understood how specific proteins move and search their target sites in the myriad of DNA sequence repertoires. We use λ-exonuclease as a model of nucleic acid-molecular motors that processively translocates along the DNA. Here, we combined single molecule FRET and molecular dynamics (MD) simulation to examine how the dynamic interaction translates into overall enzymatic activity. Transient coupling between λ-exonuclease and its substrate DNA significantly alters its translocation by a factor of ∼30 due to chemical friction between a positive reside (ARG45) of the protein and electrostatic potential (EP) along the minor groove of DNA. Repulsive interaction gives rise to futile slippage events whereas attractive coupling between ARG45 and adenines at the minor grooves provides chemical ratcheting for unidirectional translocation, preventing diffusive backtracking by electrostatic friction. We propose an anti-friction-based ratchet for processive translocation. Our study provides new insights into not only interplay between dynamic chemophysical interaction and enzyme activity but also a role of the minor groove in regulating enzymatic activity based on DNA sequences.

Heterogeneous migration routes of DNA triplet repeat slip-outs

It is unclear how the length of a repetitive DNA tract determines the onset and progression of repeat expansion diseases, but the dynamics of secondary DNA structures formed by repeat sequences are believed to play an important role. It was recently shown that three-way DNA junctions containing slip-out hairpins of CAG or CTG repeats and contiguous triplet repeats in the adjacent duplex displayed single-molecule FRET (smFRET) dynamics that were ascribed to both local conformational motions and longer-range branch migration. Here we explore these so-called "mobile" slip-out structures through a detailed kinetic analysis of smFRET trajectories and coarse-grained modeling. Despite the apparent structural simplicity, with six FRET states resolvable, most smFRET states displayed biexponential dwell-time distributions, attributed to structural heterogeneity and overlapping FRET states. Coarse-grained modeling for a (GAC)10 repeat slip-out included trajectories that corresponded to a complete round of branch migration; the structured free energy landscape between slippage events supports the dynamical complexity observed by smFRET. A hairpin slip-out with 40 CAG repeats, which is above the repeat length required for disease in several triplet repeat disorders, displayed smFRET dwell times that were on average double those of 3WJs with 10 repeats. The rate of secondary-structure rearrangement via branch migration, relative to particular DNA processing pathways, may be an important factor in the expansion of triplet repeat expansion diseases.

Multi-parameter photon-by-photon hidden Markov modeling

Single molecule Förster resonance energy transfer (smFRET) is a unique biophysical approach for studying conformational dynamics in biomacromolecules. Photon-by-photon hidden Markov modeling (H2MM) is an analysis tool that can quantify FRET dynamics of single biomolecules, even if they occur on the sub-millisecond timescale. However, dye photophysical transitions intertwined with FRET dynamics may cause artifacts. Here, we introduce multi-parameter H2MM (mpH2MM), which assists in identifying FRET dynamics based on simultaneous observation of multiple experimentally-derived parameters. We show the importance of using mpH2MM to decouple FRET dynamics caused by conformational changes from photophysical transitions in confocal-based smFRET measurements of a DNA hairpin, the maltose binding protein, MalE, and the type-III secretion system effector, YopO, from Yersinia species, all exhibiting conformational dynamics ranging from the sub-second to microsecond timescales. Overall, we show that using mpH2MM facilitates the identification and quantification of biomolecular sub-populations and their origin.

Simultaneous analyses of fluorescence decay and anisotropy decay in green fluorescent protein dimer from jellyfish Clytia gregaria: FRET and molecular dynamics simulation

Structural and dynamic behaviors of the green fluorescent protein dimer from jellyfish Clytia gregaria (cgGFP) were investigated by means of molecular dynamics (MD) simulation. Both neutral and ionic forms of the chromophore, p-hydroxybenzylideneimidazolinone (GYS) were considered. The partial atomic charges of the chromophore were derived by BCC and RESP approaches. The structures were compared between the anionic and neutral cgGFP, and between the two subunits (Sub A and Sub B) of the protein dimer. The observed fluorescence intensity and anisotropy decays were further analyzed with theoretical expressions by employing the atomic coordinates of neutral cgGFP obtained by MD simulation. It was assumed that the fluorescence quenching of GYSA and GYSB is ascribed to HB formations between heteroatoms of GYSs and nearby amino acids. Excellent agreement between the observed and calculated intensity decays, and the observed and calculated anisotropy decays were obtained with RESP1 model. The agreements were better in RESP model than those in BCC one. Mean quenching constants of GYSA and GYSB were 0.27 and 0.59 ns−1 overall MD snapshots with RESP1. Mean value of square of direction cosine between the two transition moments of GYSs was 0.74, and that of square of orientation factor was 0.53, and the FRET rates from GYSA to GYSB, and from GYSB to GYSA were 0.87 and 1.87 ns−1.

Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis.

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately 20 nm to accommodate large tRNA substrates at the A-, P- and E-sites. Consequently, the ribosome dynamics are naturally de-phased quickly. Single molecule method can detect each ribosome separately and distinguish inhomogeneous populations, which is essential to reveal the complicated mechanisms of multi-component systems. We report the details of a smFRET method based on the Nikon Ti2 inverted microscope to probe the ribosome dynamics between the ribosomal protein L27 and tRNAs. The L27 is labeled at its unique Cys 53 position and reconstituted into a ribosome that is engineered to lack L27. The tRNA is labeled at its elbow region. As the tRNA moves to different locations inside the ribosome during the elongation cycle, such as pre- and post- translocation, the FRET efficiencies and dynamics exhibit differences, which have suggested multiple subpopulations. These subpopulations are not detectable by ensemble methods. The TIRF-based smFRET microscope is built on a manual or motorized inverted microscope, with home-built laser illumination. The ribosome samples are purified by ultracentrifugation, loaded into a home-built multi-channel sample cell and then illuminated via an evanescent laser field. The reflection laser spot can be used to achieve feedback control of perfect focus. The fluorescence signals are separated by a motorized filter-turret and collected by two digital CMOS cameras. The intensities are retrieved via the NIS-Elements software.

The ribosome modulates folding inside the ribosomal exit tunnel

Proteins commonly fold co-translationally at the ribosome, while the nascent chain emerges from the ribosomal exit tunnel. Protein domains that are sufficiently small can even fold while still located inside the tunnel. However, the effect of the tunnel on the folding dynamics of these domains is not well understood. Here, we combine optical tweezers with single-molecule FRET and molecular dynamics simulations to investigate folding of the small zinc-finger domain ADR1a inside and at the vestibule of the ribosomal tunnel. The tunnel is found to accelerate folding and stabilize the folded state, reminiscent of the effects of chaperonins. However, a simple mechanism involving stabilization by confinement does not explain the results. Instead, it appears that electrostatic interactions between the protein and ribosome contribute to the observed folding acceleration and stabilization of ADR1a.

Visualizing Dynamic Processes with rapidFLIMHiRes: Ultra Fast Flim with Outstanding 10 PS Time Resolution

Fluorescence Lifetime Imaging (FLIM) has become an essential tool in Life Sciences over the last decade. However, up to now, users had to choose between high timing precision or fast data acquisition when using Time-Correlated Single Photon Counting (TCSPC) electronics. This was a drawback when investigating fast processes in cells or tissues such as protein interactions, FRET dynamics, or chemical reactions.

High force catch bond mechanism of bacterial adhesion in the human gut

Bacterial colonization of the human intestine requires firm adhesion of bacteria to insoluble substrates under hydrodynamic flow. Here we report the molecular mechanism behind an ultrastable protein complex responsible for resisting shear forces and adhering bacteria to cellulose fibers in the human gut. Using single-molecule force spectroscopy (SMFS), single-molecule FRET (smFRET), and molecular dynamics (MD) simulations, we resolve two binding modes and three unbinding reaction pathways of a mechanically ultrastable R. champanellensis (Rc) Dockerin:Cohesin (Doc:Coh) complex. The complex assembles in two discrete binding modes with significantly different mechanical properties, with one breaking at ~500 pN and the other at ~200 pN at loading rates from 1-100 nN s−1. A neighboring X-module domain allosterically regulates the binding interaction and inhibits one of the low-force pathways at high loading rates, giving rise to a catch bonding mechanism that manifests under force ramp protocols. Multi-state Monte Carlo simulations show strong agreement with experimental results, validating the proposed kinetic scheme. These results explain mechanistically how gut microbes regulate cell adhesion strength at high shear stress through intricate molecular mechanisms including dual-binding modes, mechanical allostery and catch bonds.

Inter-domain dynamics in the chaperone SurA and multi-site binding to its outer membrane protein clients

The periplasmic chaperone SurA plays a key role in outer membrane protein (OMP) biogenesis. E. coli SurA comprises a core domain and two peptidylprolyl isomerase domains (P1 and P2), but its mechanisms of client binding and chaperone function have remained unclear. Here, we use chemical cross-linking, hydrogen-deuterium exchange mass spectrometry, single-molecule FRET and molecular dynamics simulations to map the client binding site(s) on SurA and interrogate the role of conformational dynamics in OMP recognition. We demonstrate that SurA samples an array of conformations in solution in which P2 primarily lies closer to the core/P1 domains than suggested in the SurA crystal structure. OMP binding sites are located primarily in the core domain, and OMP binding results in conformational changes between the core/P1 domains. Together, the results suggest that unfolded OMP substrates bind in a cradle formed between the SurA domains, with structural flexibility between domains assisting OMP recognition, binding and release.

Blocking the Formation of Zn2+/Dye Complexes in Dye-Sensitized Solar Cells by Inserting CdS Quantum Dots into Sandwich Layer

ZnO NRAs are grown on ITO substrates by a simple chemical method. CdS QDs were deposited on ZnO NRAs by SILAR. N719 was synthesized by dipping method. J–V analysis indicates that by inserting a layer of CdS QDs, the conversion efficiency of DSSCs was improved obviously. The device with CdS QDs shows the higher conversion efficiency due to the three reasons: (1) CdS QDs enhanced adsorption spectra of DSSCs in the visible region; (2) CdS QDs block the formation of Zn2+/dye complex, it is beneficial for electros transport from dye to ZnO photoanode. It is the key to obtain higher conversion efficiency; (3) FRET dynamics exists by the introduction of CdS QDs.

Studying Structural Dynamics of Potassium Channels by Single-Molecule FRET.

Single-molecule FRET (smFRET) can visualize conformational dynamics of individual ion channels in lipid bilayers of defined composition. Dynamic and distance measurements from smFRET, combined with single channel recordings, can provide previously unattainable direct mechanistic insights into ion channel function and modulation. smFRET measurements require site-specific fluorophore labeling between two distinct sites, which is a major challenge for multimeric ion channels. This chapter aims to provide a step-by-step protocol: (1) to design concatemeric constructs with only two cysteine residues within a homotetrameric channel; (2) to express, purify, label, and reconstitute channel proteins; (3) to perform smFRET imaging on channel proteins in liposomes with an objective-based Total Internal Reflection (TIRF) microscope; and finally (4) to analyze the FRET distributions and dynamics that reflect the dynamic conformational transitions of ion channels in membranes.

Conformational transition of FGFR kinase activation revealed by site-specific unnatural amino acid reporter and single molecule FRET

Protein kinases share significant structural similarity; however, structural features alone are insufficient to explain their diverse functions. Thus, bridging the gap between static structure and function requires a more detailed understanding of their dynamic properties. For example, kinase activation may occur via a switch-like mechanism or by shifting a dynamic equilibrium between inactive and active states. Here, we utilize a combination of FRET and molecular dynamics (MD) simulations to probe the activation mechanism of the kinase domain of Fibroblast Growth Factor Receptor (FGFR). Using genetically-encoded, site-specific incorporation of unnatural amino acids in regions essential for activation, followed by specific labeling with fluorescent moieties, we generated a novel class of FRET-based reporter to monitor conformational differences corresponding to states sampled by non phosphorylated/inactive and phosphorylated/active forms of the kinase. Single molecule FRET analysis in vitro, combined with MD simulations, shows that for FGFR kinase, there are populations of inactive and active states separated by a high free energy barrier resulting in switch-like activation. Compared to recent studies, these findings support diversity in features of kinases that impact on their activation mechanisms. The properties of these FRET-based constructs will also allow further studies of kinase dynamics as well as applications in vivo.

Amyloid beta peptides inside a reconstituted cell-like liposomal system: aggregation, FRET, fluorescence oscillations and solvation dynamics.

Aggregations of amyloid-beta (Aβ) peptides were studied inside a reconstituted cell like liposomal system using time-resolved confocal microscopy. Fluorescence correlation spectroscopy (FCS) and confocal images indicate that Aβ forms a very large aggregate in bulk and more efficiently, in the bilayer region of the liposome, respectively. The aggregates formed inside the liposome gradually migrate out to bulk water. FRET, from HiLyte Fluor 488 (covalently attached to an Aβ peptide) to TRITC (tetramethylrhodamine isothiocyanate) covalently attached to a DHPE lipid present in the bilayer, reveals intermittent oscillations in the time scale of ∼0.5 s. This is attributed to the structural fluctuations of the membrane of the liposome. The solvation dynamics of Aβ in monomer and in oligomeric state is studied by monitoring the emission of HiLyte Fluor 488. The solvation dynamics of the Aβ monomer is similar to that of oligomeric aggregates in the liposome.

Conformational Fluctuations as an Intrinsic Mechanism of Action of Lipase Foldase Studied by the High-Precision FRET Toolkit and MD Simulations

The lipase foldase (Lif) of P. aeruginosa is a steric chaperon, which acts as a conformational switch for the activation of lipase A (LipA). In the Lif:LipA complex, Lif forms flexible α-helical scaffold embracing LipA in headphone-like structure. In the unbound form, Lif does not stay in the hollow “headphone” conformation but rather exhibits large-scale conformational dynamics, where this α-helical structure undergoes reversible collapses and extensions on the microsecond to sub-microsecond timescale. This process allows Lif to bind LipA despite the fickleness of structure. Structural details of the process are studied by the fluorescence spectroscopic toolkit: a FRET distance landscape is investigated by ensemble lifetime-resolved FRET (eTCSPC), the timescales of dynamics by correlation techniques (filtered FCS) and overall FRET populations and dynamics by single-molecule MFD studies. The combination of these techniques allows drawing a kinetic scheme within the limiting states of the conformational ensemble of Lif, unraveling the mechanism of Lif action. The study is supported by all-atom MD simulations. The combination of accurate FRET, MFD, FRET positioning and screening (FPS) and fFCS with MD simulations in a hybrid approach provides in-depth insight in the complexity of the conformational dynamics of flexible proteins and protein complexes.

Structural Origin of Landscape Roughness in Protein Folding from Single-Molecule FRET and All-Atom Molecular Dynamics Simulations

Folding of most single-domain proteins has been successfully described by diffusion on a one-dimensional (1D) free energy surface. Although the 1D surface is smooth, there are many local minima in the underlying energy landscape, giving rise to landscape “roughness”. According to Kramers’ reaction-rate theory, roughness slows folding kinetics by reducing the diffusion coefficient at the top of the free energy barrier that separates folded and unfolded states. By measuring the transition-path time (tTP) from a maximum likelihood analysis of photon trajectories in single molecule FRET experiments, we have recently shown that the Kramers diffusion coefficient for a designed α-helical protein, α3D, is markedly reduced (Chung and Eaton, Nature,2013). To discover the structural origin of this slow diffusion, we have combined additional single-molecule FRET measurements with all-atom molecular dynamics (MD) calculations. α3D contains 12 negatively-charged and 10 positively-charged side-chains. Analysis of the transition paths in the MD simulations shows that many non-native salt-bridges form during the folding transition path, suggesting them as the structural origin of long tTP. To test this idea, we lowered the pH to neutralize the carboxylates and eliminate salt-bridges, which increased the folding rate by about 10-fold and significantly reduced tTP. Although it was only possible to determine an upper bound for tTP, even at the highest possible solvent viscosity (15 cP), simulations of photon trajectories suggested that most, if not all, of the increase in folding rate could be accounted for by a decreased tTP and an increased Kramers diffusion coefficient. Neutralizing the carboxylates in MD simulations also increases the folding rate and diffusion coefficient and decreases tTP. These results provide the first quantitative glimpse of the effect of specific intra-molecular interactions on barrier crossing dynamics in protein folding.

Dynamics Measurement of Diffusing Single-Molecule with Tens of Millisecond Observation Time by Liposome Tethering

Single-molecule fluorescence resonance energy transfer (smFRET) is one of the most powerful techniques for deciphering the dynamics of unsynchronized biomolecules. However, smFRET is limited in its temporal resolution for the observation of the real-time dynamics of a single molecule. Here, we report a novel method for observing real-time FRET dynamics with sub-millisecond resolution by tethering FRET molecules to freely diffusing 100-nm-sized liposomes. The observation time for a single diffusing molecule was extended to 100 ms with a sub-millisecond temporal FRET resolution, which allowed for direct analysis of the transition states based on the FRET time trace using hidden Markov modeling. Using this method, we measured transition rates of up to 1500 s-1 (∼ 0.7-ms dwell time) between two conformers of a Holliday junction from the transition-density plot. The rapid diffusional migration of dimeric SSB from Deinococcus radiodurans on single-stranded (ss) DNA was directly observed in the FRET time traces at room temperature; this diffusion is faster than that of the tetrameric E. coli SSB by more than an order of magnitude. This result suggests that the diffusion of SSB on ssDNA is a general phenomenon. These results clearly demonstrate that our approach is a powerful method for studying the conformational dynamics and movements of biomolecules at sub-millisecond time scales.

Nucleosome Dynamics Studied by Single-Pair FRET and Computer Simulations

DNA in nucleosomes is sterically occluded and nucleosomes must open to allow full DNA access. We studied this process by single pair FRET (spFRET), all-atom and coarse-grained molecular dynamics. spFRET shows evidence for a new structural intermediate preceding histone dissociation, in which the (H3-H4)2 tetramer/(H2A-H2B) dimer interface is split open. This is followed by H2A-H2B dimer release from the DNA and, lastly, (H3-H4)2 tetramer removal. This open intermediate state could be demonstrated in Xenopus and yeast nucleosomes; histone variants such as H2A.Z change the mechanism of opening. We estimate that the open state is populated at 0.2 - 3 % under physiological conditions, and could have significant in vivo implications for factor-mediated histone exchange and for DNA accessibility. To look for subpopulations and changes in FRET distances during the salt-induced transition for the various FRET pairs, we used photon distribution analysis (PDA). We observed two to three populations for all the FRET pairs, their calculated distance corresponding to the values expected from the dye positions in the crystal structure. Histone tail acetylation increased these distances and shifted the midpoint of the opening transition to lower salt concentrations. The effect of histone tail modification was studied by all-atom MD simulations with 100 ns trajectories of the full nucleosome. We find evidence for an internal allosteric transition at the H2A-H3 interface induced by the removal of either the H3 or the H2A N-terminal tail. DNA dynamics on the histone core are studied by a new coarse-grained model; these simulations show a strong influence of the presence of the histone tails on DNA unwrapping, and an open state that can be stabilized by the displacement of the H3 tail into the gap between the DNA and the histone core.

Combining MFD and PIE for Accurate Single‐Pair Förster Resonance Energy Transfer Measurements

Single-pair Förster resonance energy transfer (spFRET) experiments using single-molecule burst analysis on a confocal microscope are an ideal tool to measure inter- and intramolecular distances and dynamics on the nanoscale. Different techniques have been developed to maximize the amount of information available in spFRET burst analysis experiments. Multiparameter fluorescence detection (MFD) is used to monitor a variety of fluorescence parameters simultaneously and pulsed interleaved excitation (PIE) employs direct excitation of the acceptor to probe its presence and photoactivity. To calculate accurate FRET efficiencies from spFRET experiments with MFD or PIE, several calibration measurements are usually required. Herein, we demonstrate that by combining MFD with PIE information regarding all calibration factors as well as an accurate determination of spFRET histograms can be performed in a single measurement. In addition, the quality of overlap of the different detection volumes as well as the detection of acceptor photophysics can be investigated with MFD-PIE. Bursts containing acceptor photobleaching can be identified and excluded from further investigation while bursts that contain FRET dynamics are unaffected by this analysis. We have employed MFD-PIE to accurately analyze the effects of nucleotides and substrate on the interdomain separation in DnaK, the major bacterial heat shock protein 70 (Hsp70). The interdomain distance increases from 47 Å in the ATP-bound state to 84 Å in the ADP-bound state and slightly contracts to 77 Å when a substrate is bound. This is in contrast to what was observed for the mitochondrial member of the Hsp70s, Ssc1, supporting the notion of evolutionary specialization of Hsp70s for different cellular functions in different organisms and cell organelles.

Nucleosome Dynamics Studied by Single Pair FRET and Computer Simulations

DNA in nucleosomes is sterically occluded and nucleosomes must open to allow full DNA access. We studied this process by single pair FRET (spFRET) and coarse-grained molecular dynamics. spFRET shows evidence for a new structural intermediate preceding histone dissociation, in which the (H3-H4)2 tetramer/(H2A-H2B) dimer interface is split open. This is followed by H2A-H2B dimer release from the DNA and, lastly, (H3-H4)2 tetramer removal. This open intermediate state could be demonstrated in Xenopus and yeast nucleosomes; histone variants such as H2A.Z may change the mechanism of opening.

Abstract 497: Mitochondria-targeting photosensitizers activable by two-photon absorption

Abstract Two-photon absorption (TPA) is a non-linear optical process, used to activate photosensitizers in the NIR region (700 - 850 nm) thus affording greater tissue penetration in photodynamic therapy (PDT). Since the efficiency of this process has a quadratic dependence on the light intensity, the photodynamic effects are restricted to a small area around the laser's focal point, thus providing spatial selectivity. Although most clinically approved photosensitizers can be activated by TPA, they generally have very small cross-sections (generally below 10 GM around 800 nm). Thus, light intensities required to activate them could damage normal surrounding tissues. The TPA of photosensitizers can however be improved, without perturbing their ability to generate singlet oxygen, by conjugating them to effective TPA chromophores. These then serve as TPA antennae and upon activation transfer their energy to the photosensitizer which then generates singlet oxygen for PDT. In this study a porphyrin-rhodamine B dyad (TPP-Rh), previously demonstrated to target mitochondria, was evaluated as a two-photon photosensitizer for PDT. TPP-Rh, was synthesized by conjugating a monohydroxy porphyrin photosensitizer (TPP-OH) to rhodamine B (Rh B), an effective TPA chromophore, via a saturated hydrocarbon linker, thus enhancing intramolecular FRET between the dyad components and ultimately the photosensitizer's TPA. To evaluate the efficiency of TPP-Rh as a two-photon photosensitizer we studied both its one and two-photon absorption photophysical properties in comparison to those of its individual components: Rh B and TPP-OH. These included: FRET kinetics, FRET dynamics, and the singlet oxygen generation kinetics. A FRET rate of 1.679 × 1012 s−1 and a 99 % FRET efficiency was calculated for TPP-Rh from its spectral properties suggesting efficient FRET from the TPA moiety (Rh B) to the photosensitizer moiety (TPP). In addition to this, TPP-Rh produced singlet oxygen at comparable rates when excited with both TPA at 800 nm and one photon absorption (OPA) at 650 nm. Thus, this indicates that conjugating photosensitizers to rhodamine B could provide several advantages in PDT: deeper tissue penetration, spatial selectivity and selective photosensitizer accumulation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 497.