FRET Efficiency Research Articles

Flow cytometry FRET reveals post-translational modifications drive Protein Phosphatase-5 conformational changes in mammalian cells.

The serine/threonine Protein Phosphatase-5 (PP5) plays an essential role in regulating hormone and stress-induced signaling networks as well as extrinsic apoptotic pathways in cells. Unlike other Protein Phosphatases, PP5 possesses both regulatory and catalytic domains, and its function is further modulated through post-translational modifications (PTMs). PP5 contains a tetratricopeptide repeat (TPR) domain, which usually inhibits its phosphatase activity by blocking the active site (closed conformation). Certain activators bind to the PP5-TPR domain, alleviating this inhibition and allowing the catalytic domain to adopt an active (open) conformation. While this mechanism has been proposed based on structural and biophysical studies, PP5 conformational changes and activity have yet to be observed in cells. Here, we designed and developed a flow cytometry-based fluorescence resonance energy transfer (FC-FRET) method, enabling real-time observation of PP5 autoinhibition and activation within live mammalian cells. By quantifying FRET efficiency using sensitized emission, we established a standardized and adaptable data acquisition workflow. Our findings revealed that, in a cellular context, PP5 exists in multiple conformational states, none of which alone fully predicts its activity. Additionally, we have demonstrated that PTMs such as phosphorylation and SUMOylation impact PP5 conformational changes, representing a significant advancement in our understanding of its regulatory mechanisms.

DNA Origami Vesicle Sensors with Triggered Single-Molecule Cargo Transfer.

Interacting with living systems typically involves the ability to address lipid membranes of cellular systems. The first step of interaction of a nanorobot with a cell will thus be the detection of binding to a lipid membrane. Utilizing DNA origami, we engineered a biosensor with single-molecule Fluorescence Resonance Energy Transfer (smFRET) as transduction mechanism for precise lipid vesicle detection and cargo delivery. The system hinges on a hydrophobic ATTO647N modified single-stranded DNA (ssDNA) leash, protruding from a DNA origami nanostructure. In a vesicle-free environment, the ssDNA coils, ensuring high FRET efficiency. Upon vesicle binding to cholesterol anchors on the DNA origami, hydrophobic ATTO647N induces the ssDNA to stretch towards the lipid bilayer, reducing FRET efficiency. As the next step, the sensing strand serves as molecular cargo that can be transferred to the vesicle through a triggered strand displacement reaction. Depending on the number of cholesterols on the displacer strands, we either induce a diffusive release of the fluorescent load towards neighboring vesicles or a stoichiometric release of a single cargo-unit to the vesicle on the nanosensor. Ultimately, our multi-functional liposome interaction and detection platform opens up pathways for innovative biosensing applications and controllable stoichiometric loading of vesicles with single-molecule control.

Single-Molecule Characterization of Heterogeneous Oligomer Formation during Co-Aggregation of 40- and 42-Residue Amyloid-β.

The two most abundant isoforms of amyloid-β (Aβ) are the 40- (Aβ40) and 42-residue (Aβ42) peptides. Since they coexist and there is a correlation between toxicity and the ratio of the two isoforms, quantitative characterization of their interactions is crucial for understanding the Aβ aggregation mechanism. In this work, we follow the aggregation of individual isoforms in a mixture using single-molecule FRET spectroscopy by labeling Aβ42 and Aβ40 with the donor and acceptor fluorophores, respectively. We found that there are two phases of aggregation. The first phase consists of coaggregation of Aβ42 with a small amount of Aβ40, while the second phase results mostly from aggregation of Aβ40. We also found that the aggregation of Aβ42 is slowed by Aβ40 while the aggregation of Aβ40 is accelerated by Aβ42 in a concentration-dependent manner. The formation of oligomers was monitored by incubating mixtures in a plate reader and performing a single-molecule free-diffusion experiment at several different stages of aggregation. The detailed properties of the oligomers were obtained by maximum likelihood analysis of fluorescence bursts. The FRET efficiency distribution is much broader than that of the Aβ42 oligomers, indicating the diversity in isoform composition of the oligomers. Pulsed interleaved excitation experiments estimate that the fraction of Aβ40 in the co-oligomers in a 1:1 mixture of Aβ42 and Aβ40 varies between 0 and 20%. The detected oligomers were mostly co-oligomers especially at the physiological ratio of Aβ42 and Aβ40 (1:10), suggesting the critical role of Aβ40 in oligomer formation and aggregation.

FRET calibration by symmetrization on the cell surface

Maximum position of Shannon-information of FRET frequency distribution represented as a function of the α (or G) calibrating factor as calculated in the framework of dual-laser flow cytometric FRET method – also called 3-cube FRET – gives a fairly good approximation of the real α, using only a single FRET sample. The estimation is accurate for completely symmetric FRET distributions like Gaussian and uniform. For non-symmetric distributions, the committed error has been found to depend on the direction and degree of deviation from the complete symmetry. The maximum information is characterized also by zero skewness, and minima in kurtosis and in the χ-value, defined as the “absolute deviation from the Gaussian”, as other α- functions characterizing shape. As to practice, information maximum of FRET distribution can be used to determine α, whenever symmetry can be assumed. In other cases, the goodness of estimation can be inferred from the information content and shape parameters (skewness, kurtosis and “χ-value”) of the primarily measured FRET parameter A′, based on their inverse correlations with their counterparts computed with the target FRET distribution. Above a threshold in FRET efficiency or its coefficient of variation, resonant peaks (“Ω-curves”) in FRET information appear on the α-scale, with their positions stabilized around α values with reduced fluctuations dictated by symmetry of the target FRET distribution. The phenomenon is explained by a FRET-induced broadening of the A′ distribution reaching the domain containing A′ = −α.

Abstract Mo069: Phospholamban Acetylation Enhances Cardiomyocyte Calcium Cycling Under Conditions of High-Fat Feeding

Background: Diet-induced obesity (DIO) is an increasing driver of cardiovascular disease and cardiac dysfunction. The breakdown of excessive fat and sugar intake increases the presence of intracellular acetyl-CoA, which promotes the acetylation of available lysines, altering the stability and activity of these proteins. While numerous proteins involved in cardiac health have been identified as targets of acetylation, the impact of acetylation on calcium handling sarcoplasmic reticulum (SR) proteins, particularly the SERCA2a regulator phospholamban, remains poorly understood. Hypothesis: Phospholamban acetylation is upregulated by exposure to high fat conditions, in turn driving SERCA2a activity and promoting faster calcium SR reuptake. Approach: Left ventricle (LV) tissue from DIO mice and controls, and human LV tissue from control and diabetic patients, was collected and analyzed using immunoblotting and acetylation was quantified by immunoprecipitation with acetyl-lysine antibodies.Isolated working hearts from DIO and control mice were assessed before and after treatment with acute HDAC activator ITSA-1 and SIRT1 activator SRT1720 to promote deacetylation. Adult ventricular cardiomyocytes were isolated from DIO and control mice, and DIO cells were incubated in media containing excess lipid substrates (1% Lipid Mix 1, Millipore Sigma) and ITSA-1 orSRT1720. Changes in stimulated calcium dynamics were assessed using Fura-2AM and Fluo-4AM imaging. Acetylmimetic/acetylation incompetent PLN mutants were transfected into cultured cells to evaluate SERCA2a binding, oligomerization, and FRET efficiency. Results: Phospholamban acetylation significantly increased in LV tissue from both DIO mice and human diabetic patients. Acute treatment of isolated hearts and isolated cardiomyocytes from DIO mice with HDAC and SIRT1 activators significantly reduced contractility, relaxation, and SR calcium reuptake rates. Mutations in PLN mimicking or preventing lysine acetylation altered oligomerization and SERCA2a binding. Conclusions: Obesity increases the acetylation of phospholamban and enhances the reuptake of calcium into the SR, while deacetylation reverses these effects. These data directly tie nutritive status to a novel phospholamban post-translational modification that may directly impact myocardial calcium handling and contractile function.

Mechanistic insights from the atomic-level quaternary structure of short-lived GPCR oligomers in live cells.

The functional significance of the interactions between proteins in living cells to form short-lived quaternary structures cannot be overemphasized. Yet, quaternary structure information is not captured by current methods, neither can those methods determine structure within living cells. The dynamic versatility, abundance, and functional diversity of G protein-coupled receptors (GPCRs) pose myriad challenges to existing technologies but also present these proteins as the ideal testbed for new technologies to investigate the complex inter-regulation of receptor-ligand, receptor-receptor, and receptor-downstream effector interfaces in living cells. Here, we present development and use of a novel method capable of overcoming existing challenges by combining distributions (or spectrograms) of FRET efficiencies from populations of fluorescently tagged proteins associating into oligomeric complexes in live cells with diffusion-like trajectories of FRET donors and acceptors obtained from molecular dynamics (MD) simulations. Our approach provides an atom-level picture of the binding interfaces within oligomers of the human secretin receptor (hSecR) in live cells and allows for extraction of mechanistic insights into the function of GPCRs oligomerization. This FRET-MD spectrometry approach is a robust platform for investigating protein-protein binding mechanisms and opens a new avenue for investigating stable as well as fleeting quaternary structures of any membrane proteins in living cells.

Recovering true FRET efficiencies from smFRET investigations requires triplet state mitigation

Single-molecule fluorescence resonance energy transfer (smFRET) methods employed to quantify time-dependent compositional and conformational changes within biomolecules require elevated illumination intensities to recover robust photon emission streams from individual fluorophores. Here we show that outside the weak-excitation limit, and in regimes where fluorophores must undergo many rapid cycles of excitation and relaxation, non-fluorescing, excitation-induced triplet states with lifetimes orders of magnitude longer lived than photon-emitting singlet states degrade photon emission streams from both donor and acceptor fluorophores resulting in illumination-intensity-dependent changes in FRET efficiency. These changes are not commonly taken into consideration; therefore, robust strategies to suppress excited state accumulations are required to recover accurate and precise FRET efficiency, and thus distance, estimates. We propose both robust triplet state suppression and data correction strategies that enable the recovery of FRET efficiencies more closely approximating true values, thereby extending the spatial and temporal resolution of smFRET.

Single-molecule fluorescence multiplexing by multi-parameter spectroscopic detection of nanostructured FRET labels

Multiplexed, real-time fluorescence detection at the single-molecule level can reveal the stoichiometry, dynamics and interactions of multiple molecular species in mixtures and other complex samples. However, fluorescence-based sensing is typically limited to the detection of just 3–4 colours at a time due to low signal-to-noise ratio, high spectral overlap and the need to maintain the chemical compatibility of dyes. Here we engineered a palette of several dozen composite fluorescent labels, called FRETfluors, for multiplexed spectroscopic measurements at the single-molecule level. FRETfluors are compact nanostructures constructed from three chemical components (DNA, Cy3 and Cy5) with tunable spectroscopic properties due to variations in geometry, fluorophore attachment chemistry and DNA sequence. We demonstrate FRETfluor labelling and detection for low-concentration (<100 fM) mixtures of mRNA, dsDNA and proteins using an anti-Brownian electrokinetic trap. In addition to identifying the unique spectroscopic signature of each FRETfluor, this trap differentiates FRETfluors attached to a target from unbound FRETfluors, enabling wash-free sensing. Although usually considered an undesirable complication of fluorescence, here the inherent sensitivity of fluorophores to the local physicochemical environment provides a new design axis complementary to changing the FRET efficiency. As a result, the number of distinguishable FRETfluor labels can be combinatorically increased while chemical compatibility is maintained, expanding prospects for spectroscopic multiplexing at the single-molecule level using a minimal set of chemical building blocks.

Yolk-shell nanospheres based silane-functionalized carbon dots: Designed synthesis and application in highly selective determination of aflatoxin biosynthesis-related genes nor-1 in cereal crops

This study presented advanced monitoring approaches for the aflatoxin gene cluster in Aspergillus flavus and Aspergillus parasiticus, which are effective tools for controlling aflatoxin proliferation in the food chain. A novel approach involved a ratiometric fluorescence probe based on yolk-shell nanospheres (bCDs@SiO2@gQDs). The probe's fluorescence was activated by DNA hybridization with the modified bCDs@SiO2@gQDs and gold nanoparticles (AuNPs), functioning through donor–acceptor pairs. The exceptional properties of the gQDs and the high FRET efficiency of this system established a functionalized platform for DNA labeling. When the nor-1 gene was present, FRET between the gQDs and AuNPs triggered fluorescence quenching, enabling nor-1 gene detection. The yolk-shell nanostructure of the fluorescent nanoprobes provided an internal reference, reducing experimental errors due to background noise, instrumentation factors, and probe concentration. This allowed for accurate and highly sensitive quantitative determination of the nor-1 gene. The ratiometric fluorescence assay demonstrated a broad linear range (0.1–100 nM) and a low detection limit (12.83 pM) for the nor-1 gene. Tests on non-contaminated maize samples spiked with various concentrations of the nor-1 gene achieved recovery rates between 95.43 % and 101.46 %. This biosensor represents a significant advancement in food safety, offering a simple, rapid, and sensitive method for monitoring agricultural products.

NIR FRET Luminescence in Rhenium Complex and Dye Co‐Loaded Polymer Nanoparticles

AbstractPhotoactive transition‐metal complexes are luminophores combining high photostability and long luminescence lifetimes. However, reduced optical performance in aqueous solutions has limited their use in biological systems. Herein, the physicochemical and photophysical properties and bioimaging compatibility of Re diimine complexes and near‐infrared (NIR) emitting Cy5 dyes coencapsulated in polymer nanoparticles (NPs) are investigated. By varying the polymers, NPs with sizes from 20 to 70 nm and encapsulating ≤ 40 wt.% of Re complexes, i.e., ≈11 000 Re complexes per NP, are obtained. The photoluminescence (PL) quantum yields of the Re complexes increase eightfold to ≈50% upon encapsulation (vs 6–7% in acetonitrile), resulting in PL brightness up to 108 m−1 cm−1 and PL lifetimes of 3–4 µs. Coencapsulation of Cy5 yields very bright NIR emission upon Re complex excitation. Very close Re‐to‐Cy5 donor–acceptor distances down to ≤2 nm and FRET efficiencies over 90% are confirmed by PL lifetime measurements. The Re‐Cy5 NPs enter mammalian cells for high‐contrast PL imaging in both visible and NIR. This detailed characterization provides a better understanding of the photophysical properties of the transition‐metal‐dye FRET NPs and presents a vital step toward the efficient design of a new class of bright luminescent NP probes.

Single-molecule FRET and molecular diffusion analysis characterize stable oligomers of amyloid-β 42 of extremely low population

AbstractSoluble oligomers produced during protein aggregation have been thought to be toxic species causing various diseases. Characterization of these oligomers is difficult because oligomers are a heterogeneous mixture, which is not readily separable, and may appear transiently during aggregation. Single-molecule spectroscopy can provide valuable information by detecting individual oligomers, but there have been various problems in determining the size and concentration of oligomers. In this work, we develop and use a method that analyzes single-molecule fluorescence burst data of freely diffusing molecules in solution based on molecular diffusion theory and maximum likelihood method. We demonstrate that the photon count rate, diffusion time, population, and Förster resonance energy transfer (FRET) efficiency can be accurately determined from simulated data and the experimental data of a known oligomerization system, the tetramerization domain of p53. We used this method to characterize the oligomers of the 42-residue amyloid-β (Aβ42) peptide. Combining peptide incubation in a plate reader and single-molecule free-diffusion experiments allows for the detection of stable oligomers appearing at various stages of aggregation. We find that the average size of these oligomers is 70-mer and their overall population is very low, less than 1 nM, in the early and middle stages of aggregation of 1 µM Aβ42 peptide. Based on their average size and long diffusion time, we predict the oligomers have a highly elongated rod-like shape.

Essential role of momentum-forbidden dark excitons in the energy transfer responses of monolayer transition-metal dichalcogenides

We present a theoretical investigation of exciton-mediated Förster resonant energy transfers (FRET’s) from photoexcited quantum dots (QD’s) to transition-metal dichalcogenide monolayers (TMD-ML’s), implemented by the quantum theory of FRET on the base of first-principles-calculated exciton fine structures. With the enhanced electron-hole Coulomb interactions, atomically thin TMD-MLs are shown to serve as an exceptional platform for FRET that are mediated purely by excitons and take full advantage of the superior excitonic properties. Remarkably, the energy-transfer responses of atomically thin TMD-ML’s are shown to be dictated by the momentum-forbidden dark excitons rather than the commonly recognized bright ones. Specifically, the longitudinal dark exciton states following the exchange-driven light-like linear band dispersion play a key role in grading up the efficiency and robustness of FRET of TMD-ML against the inhomogeneity of QD-donor ensembles. With the essential involvement of dark excitons, the FRET responses of TMD-ML’s no longer follow the distance power law as classically predicted and, notably, cannot manifest the dimensionality of the donor-acceptor system.

Enhanced imaging of protein-specific palmitoylation with HCR-based cis-membrane multi-FRET

Palmitoylation plays an important role in modulating protein trafficking, stability, and activity. The major predicament in protein palmitoylation study is the lack of specific and sensitive tools to visualize protein-specific palmitoylation. Although FRET approach was explored by metabolically labeled palmitic acid and antibody recognized target protein. The trans-membrane strategy suffers from low FRET efficiency due to the donor and acceptor located at different sides of membrane. Herein, we proposed a cis-membrane multi–fluorescence resonance energy transfer (multi–FRET) for amplified visualization of specific palmitoylated proteins through metabolic labeling and targeted recognition. The azido-palmitic acid (azido-PA) was metabolically incorporated into cellular palmitoylated proteins, followed by reacting with dibenzylcylooctyne-modified Cy5 (DBCO-Cy5) through copper-free click chemistry. The protein probe was attached to targeted protein by specific peptide recognition, which initiates a hybridization chain reaction (HCR) amplification process. The cis-membrane labeling method enables effective intramolecular donor-acceptor distance and allow to increase FRET efficiency. Simultaneously, HCR amplification triggered multi–FRET phenomenon with significantly improved FRET efficiency. With the superiority, this strategy has achieved the enhanced FRET imaging of palmitoylated PD-L1 and visualizing the palmitoylation changes of on PD-L1 under drug treatment. Furthermore, the established method successfully amplified visualization of PD-L1 palmitoylation in vivo and mice tumor slice. We envision the approach would provide a useful platform to investigate the effects of palmitoylation on the protein structure and function.

Aggregation enhanced FRET: A simple but efficient strategy for the ratiometric detection of uranyl ion

Uranium is one of the most important radionuclides but could also cause potential health risks to human beings due to its radioactive and chemical toxicity. It is an urgent task to develop a simple but efficient sensing platform for UO22+, the main existing form of uranium in environment. Herein, a rhodamine-functionalized carbon dots (o-CDs-Rho) was synthesized and applied for UO22+ sensing through a simple but novel aggregation-enhanced FRET strategy. The weak FRET efficiency (16.2%) of o-CDs-Rho in dispersed solution is significantly enhanced (>77.2%) after UO22+ triggered aggregation due to the increased number of rhodamine acceptors around each CDs from dispersed 80 to aggregated 2800. This is the first ratiometric fluorescence sensor with an inverse change of fluorescence intensity at dual emission wavelengths under single-wavelength excitation for UO22+. Under optimized experiment conditions, o-CDs-Rho nanosensor shows a low detection limit of 53 nM and excellent selectivity. Meanwhile, the as-prepared nanosensor also shows high reliability and stability. These excellent properties make it successful in detecting uranium content in real samples.

Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins

Single-molecule Förster-resonance energy transfer (smFRET) experiments allow the study of biomolecular structure and dynamics in vitro and in vivo. We performed an international blind study involving 19 laboratories to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems with distinct conformational changes and dynamics, we obtained an uncertainty of the FRET efficiency ≤0.06, corresponding to an interdye distance precision of ≤2 Å and accuracy of ≤5 Å. We further discuss the limits for detecting fluctuations in this distance range and how to identify dye perturbations. Our work demonstrates the ability of smFRET experiments to simultaneously measure distances and avoid the averaging of conformational dynamics for realistic protein systems, highlighting its importance in the expanding toolbox of integrative structural biology.

In situ FRET-based localization of the N terminus of myosin binding protein-C in heart muscle cells

Cardiac myosin binding protein-C (cMyBP-C) is a thick filament-associated regulatory protein frequently found mutated in patients suffering from hypertrophic cardiomyopathy (HCM). Recent invitro experiments have highlighted the functional significance of its N-terminal region (NcMyBP-C) for heart muscle contraction, reporting regulatory interactions with both thick and thin filaments. To better understand the interactions of cMyBP-C in its native sarcomere environment, insitu Foerster resonance energy transfer-fluorescence lifetime imaging(FRET-FLIM) assays were developed to determine the spatial relationship between the NcMyBP-C and the thick and thin filaments in isolated neonatal rat cardiomyocytes (NRCs). Invitro studies showed that ligation of genetically encoded fluorophores to NcMyBP-C had no or little effect on its binding to thick and thin filament proteins. Using this assay, FRET between mTFP conjugated to NcMyBP-C and Phalloidin-iFluor 514 labeling the actin filaments in NRCs was detected by time-domain FLIM. The measured FRET efficiencies were intermediate between those observed when the donor was attached to the cardiac myosin regulatory light chain in the thick filaments and troponin T in the thin filaments. These results are consistent with the coexistence of multiple conformations of cMyBP-C, some with their N-terminal domains binding to the thin filament and others binding to the thick filament, supporting the hypothesis that the dynamic interchange between these conformations mediates interfilament signaling in the regulation of contractility. Moreover, stimulation of NRCs with β-adrenergic agonists reduces FRET between NcMyBP-C and actin-bound Phalloidin, suggesting that cMyBP-C phosphorylation reduces its interaction with the thin filament.

FRET–Calc: A free software and web server for Förster Resonance Energy Transfer Calculation

Förster Resonance Energy Transfer Calculator (FRET−Calc) is a program and web server that analyzes molar extinction coefficient of the acceptor, emission spectrum of the donor, and the refractive index spectrum of the donor/acceptor blend. Its main function is to obtain important parameters of the FRET process from experimental data, such as: (i) effective refractive index, (ii) overlap integral, (iii) Förster radius, (iii) FRET efficiency and (iv) FRET rate. FRET−Calc is license free software that can be run via dedicated web server (nanocalc.org) or downloading the program executables (for Unix, Windows, and macOS) from the FRET−Calc repository on GitHub. The program features a user−friendly interface, making it suitable for materials research and teaching purposes. In addition, the program is optimized to run on normal computers and is lightweight. An example will be given with the step by step of its use and results obtained.

Rapid and ultrasensitive miRNA detection by combining endonuclease reactions in a rolling circle amplification (RCA)-based hairpin DNA fluorescent assay.

MicroRNA (miRNA) sensing strategies employing rolling circle amplification (RCA) coupled with the hairpin DNA (HD) probe-mediated FRET assay have shown promise, but achieving rapid, sensitive, and specific detection of target miRNA remains a challenge in clinical diagnostics. Herein, we incorporate PstI endonuclease cleavage (PEC) into a conventional RCA-based HD probe FRET assay to develop an effective and feasible method. Long single-stranded RCA products are synthesized from miRNA-21 loaded on a circular dumbbell DNA, and the resultant RCA products self-assemble to generate long HD structures with double-stranded stem regions that are specifically recognized and cleaved by PstI endonucleases when incubated with PstI enzymes. This releases large amounts of short single-stranded DNA fragments that hybridize and open to the complementary loop-stem regions of HD probes labeled with FAM at one end and BHQ-1 at the other, resulting in a reduction in FRET efficiency. This assay achieves a 39.7 aM detection limit for target miRNA-21, approximately 37-fold higher than that of the conventional assay (1.5 fM). Moreover, quantitative detection is possible in a wide range from 1 aM to 1pM within 90min with high sequence specificity. We demonstrate the assay with the detection of target miRNA-21 in total RNA extracted from MCF-7 cancer cells.

From single-molecule to synchrotron: New tools for investigating the molecular mechanisms of aberrant DNA diseases.

Damage in DNA can interrupt and alter its conformational flexibility—which drives crucial site-specific functions—leading to aberrant DNA diseases. It is not fully understood exactly what role these conformations play in the processes undertaken by DNA, nor how they might be affected by various damage. Here we present an overview and comparison of two novel and emergent techniques to study such conformations; single molecule Förster resonance energy transfer (smFRET) and X-ray scattering interferometry (XSI). We are focusing on the RNaseH2 complex, mutations that are responsible for Aicardi-Goutières syndrome (AGS), to establish how the search space is minimised as it finds and cleaves RNA/DNA hybrids, and whether this is driven by conformational changes. We also introduce O6-methylguanine (O6-MeG), ribonucleotide, nicks and gaps; all prevalent modifications to DNA which without repair would lead to toxic intermediates. It is hypothesised that when the damage is present the increase in flexibility of DNA signals proteins to promote repair. smFRET was used to measure the conformational changes when the different types of damage, were present in a DNA duplex. A change in FRET efficiency is seen for all the types of damage, with the nick and gap damage having a greater impact on the mean and spread of the FRET efficiency. These results provide initial support for the hypothesis that damage alters the overall structure and dynamics of DNA duplexes and that the presence of these lesions and their respective repair mechanisms binds the DNA in varying levels of flexibility.

An integrative approach to molecular dynamics and single molecule FRET techniques.

Molecular dynamics (MD) simulation and single-molecule fluorescence resonance energy transfer (smFRET) spectroscopy are two techniques that can be used to study conformational heterogeneity of proteins. However, significant challenges exist in these techniques that limit their effectiveness. All-atom MD is currently too slow to adequately sample conformational space of proteins. From an experimental perspective, deciding where to place the fluorescent dye reporters for smFRET measurements can lead to a great deal of wasted time, effort, and expense in producing labeled proteins that may or may not provide useful information. Even if the dyes are suitably placed, interpretation of smFRET data is also challenging since many conformations can lead to the same FRET efficiency for a single smFRET-labeled protein. In this work, we have addressed these problems using a highly integrative approach to smFRET and MD. We have developed an iterative method to characterize the conformational ensemble of proteins by combining MD-based enhanced sampling techniques and smFRET spectroscopy, where the computational and experimental approaches can benefit mutually from each other. We first employed an enhanced sampling based MD method (ES-MD) using system-specific collective variables for the system under study to sample various conformations. Since these simulations are biased, the relative weight of different populations of samples may not be accurate. In the next step, we predicted the FRET efficiency distributions for a large number of residue pairs and from the imperfect results determined the residues that are predicted to provide the most informative FRET distributions. The measured smFRET data was then used within a resampling scheme to correct the original computed set of conformations, which was then used to predict additional smFRET distributions for other labeled residues. We have examined this methodology on intrinsically-disordered/structured and membrane/soluble proteins.