Increasing Signal Intensity of Fluorescent Oligo-Labeled Antibodies to Enable Combination Multiplexing.

Full-spectrum flow cytometry has increased antibody-based multiplexing, yet further increases remain potentially impactful. We recently proposed how fluorescence multiplexing using spectral imaging and combinatorics (MuSIC) could do so using tandem dyes and an oligo-based antibody labeling method. In this work, we found that such labeled antibodies had significantly lower signal intensities than conventionally labeled antibodies in human cell experiments. To improve signal intensity, we tested moving the fluorophores from the original external (ext.) 5' or 3' end-labeled orientation to internal (int.) fluorophore modifications. Cell-free spectrophotometer measurements showed a ∼6-fold signal intensity increase of the new int. configuration compared to the previous ext. configuration. Time-resolved fluorescence and fluorescence correlation spectroscopy showed that the ∼3-fold brightness difference is due to static quenching most likely by the oligo or solution in the ext. configuration. Spectral flow cytometry experiments using peripheral blood mononuclear cells show int. MuSIC probe-labeled antibodies (i) retained increased signal intensity while having no significant difference in the estimated % of CD8+ lymphocytes and (ii) labeled with Atto488, Atto647, and Atto488/647 combinations can be demultiplexed in triple-stained samples. The antibody labeling approach is general and can be broadly applied to many biological and diagnostic applications where spectral detection is available.

Ensemble Switching Unveils a Kinetic Rheostat Mechanism of the Eukaryotic Thiamine Pyrophosphate Riboswitch.

Thiamine pyrophosphate (TPP) riboswitches regulate thiamine metabolism by inhibiting the translation of enzymes essential to thiamine synthesis pathways upon binding to thiamine pyrophosphate in cells across all domains of life. Recent work on the Arabidopsis thaliana TPP riboswitch suggests a multi-step TPP binding process involving multiple riboswitch configurational ensembles and that Mg2+ dependence underlies the mechanism of TPP recognition and subsequent transition to the expression-inhibiting state of the aptamer domain followed by changes in the expression platform. However, details of the relationship between TPP riboswitch conformational changes and interactions with TPP and Mg2+ ¬¬in the aptamer domain constituting this mechanism are unknown. Therefore, we integrated single-molecule multiparameter fluorescence and force spectroscopy with atomistic molecular dynamics simulations and found that conformational transitions within the aptamer domain's sensor helices associated with TPP and Mg2+ ligand binding occurred between at least five different ensembles on timescales ranging from µs to ms. These dynamics are orders of magnitude faster than the 10 second-timescale folding kinetics associated with expression-state switching in the switch sequence. Together, our results show that a TPP and Mg2+ dependent mechanism determines dynamic configurational state ensemble switching of the aptamer domain's sensor helices that regulates the stability of the switch helix, which ultimately may lead to the expression-inhibiting state of the riboswitch. Additionally, we propose that two pathways exist for ligand recognition and that this mechanism underlies a kinetic rheostat-like behavior of the Arabidopsis thaliana TPP riboswitch.

Domain tethering impacts dimerization and DNA-mediated allostery in the human transcription factor FoxP1.

Transcription factors are multidomain proteins with specific DNA binding and regulatory domains. In the human FoxP subfamily (FoxP1, FoxP2, FoxP3, and FoxP4) of transcription factors, a 90 residue-long disordered region links a Leucine Zipper (ZIP)-known to form coiled-coil dimers-and a Forkhead (FKH) domain-known to form domain swapping dimers. We used replica exchange discrete molecular dynamics simulations, single-molecule fluorescence experiments, and other biophysical tools to understand how domain tethering in FoxP1 impacts dimerization at ZIP and FKH domains and how DNA binding allosterically regulates their dimerization. We found that domain tethering promotes FoxP1 dimerization but inhibits a FKH domain-swapped structure. Furthermore, our findings indicate that the linker mediates the mutual organization and dynamics of ZIP and FKH domains, forming closed and open states with and without interdomain contacts, thus highlighting the role of the linkers in multidomain proteins. Finally, we found that DNA allosterically promotes structural changes that decrease the dimerization propensity of FoxP1. We postulate that, upon DNA binding, the interdomain linker plays a crucial role in the gene regulatory function of FoxP1.

Unraveling multi-state molecular dynamics in single-molecule FRET experiments. II. Quantitative analysis of multi-state kinetic networks.

Single-molecule Förster Resonance Energy Transfer (smFRET) experiments are ideally suited to resolve the structural dynamics of biomolecules. A significant challenge to date is capturing and quantifying the exchange between multiple conformational states, mainly when these dynamics occur on the sub-millisecond timescale. Many methods for quantitative analysis are challenged if more than two states are involved, and the appropriate choice of the number of states in the kinetic network is difficult. An additional complication arises if dynamically active molecules coexist with pseudo-static molecules in similar conformational states with undistinguishable Förster Resonance Energy Transfer (FRET) efficiencies. To address these problems, we developed a quantitative integrative analysis framework that combines the information from FRET-lines that relate average fluorescence lifetimes and intensities in two-dimensional burst frequency histograms, fluorescence decays obtained by time-correlated single-photon-counting, photon distribution analysis of the intensities, and fluorescence correlation spectroscopy. Individually, these methodologies provide ambiguous results for the characterization of dynamics in complex kinetic networks. However, the global analysis approach enables accurate determination of the number of states, their kinetic connectivity, the transition rate constants, and species fractions. To challenge the potential of smFRET experiments for studying multi-state kinetic networks, we apply our integrative framework using a set of synthetic data for three-state systems with different kinetic connectivity and exchange rates. Our methodology paves the way toward an integrated analysis of multiparameter smFRET experiments that spans all dimensions of the experimental data. Finally, we propose a workflow for the analysis and show examples that demonstrate the usefulness of this toolkit for dynamic structural biology.

Unraveling multi-state molecular dynamics in single-molecule FRET experiments. I. Theory of FRET-lines.

Conformational dynamics of biomolecules are of fundamental importance for their function. Single-molecule studies of Förster Resonance Energy Transfer (smFRET) between a tethered donor and acceptor dye pair are a powerful tool to investigate the structure and dynamics of labeled molecules. However, capturing and quantifying conformational dynamics in intensity-based smFRET experiments remains challenging when the dynamics occur on the sub-millisecond timescale. The method of multiparameter fluorescence detection addresses this challenge by simultaneously registering fluorescence intensities and lifetimes of the donor and acceptor. Together, two FRET observables, the donor fluorescence lifetime τD and the intensity-based FRET efficiency E, inform on the width of the FRET efficiency distribution as a characteristic fingerprint for conformational dynamics. We present a general framework for analyzing dynamics that relates average fluorescence lifetimes and intensities in two-dimensional burst frequency histograms. We present parametric relations of these observables for interpreting the location of FRET populations in E-τD diagrams, called FRET-lines. To facilitate the analysis of complex exchange equilibria, FRET-lines serve as reference curves for a graphical interpretation of experimental data to (i) identify conformational states, (ii) resolve their dynamic connectivity, (iii) compare different kinetic models, and (iv) infer polymer properties of unfolded or intrinsically disordered proteins. For a simplified graphical analysis of complex kinetic networks, we derive a moment-based representation of the experimental data that decouples the motion of the fluorescence labels from the conformational dynamics of the biomolecule. Importantly, FRET-lines facilitate exploring complex dynamic models via easily computed experimental observables. We provide extensive computational tools to facilitate applying FRET-lines.

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.

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.

Structural Dynamics of Glutamate Signaling Systems by smFRET.

Single-molecule Förster resonance energy transfer (smFRET) is a powerful technique for investigating the structural dynamics of biological macromolecules. smFRET reveals the conformational landscape and dynamic changes of proteins by building on the static structures found using cryo-electron microscopy, x-ray crystallography, and other methods. Combining smFRET with static structures allows for a direct correlation between dynamic conformation and function. Here, we discuss the different experimental setups, fluorescence detection schemes, and data analysis strategies that enable the study of structural dynamics of glutamate signaling across various timescales. We illustrate the versatility of smFRET by highlighting studies of a wide range of questions, including the mechanism of activation and transport, the role of intrinsically disordered segments, and allostery and cooperativity between subunits in biological systems responsible for glutamate signaling.

[Cardiovascular effects of new non-insulinic anti-diabetes drugs]. / Efectos cardiovasculares de los nuevos fármacos no insulínicos en diabetes.

Diabetes mellitus is currently a serious public health problem worldwide, that increases the risk of presenting microvascular and macrovascular complications. Although achieving the recommended blood glucose goals reduces the risk of microvascular complications, the effect of the drugs used to treat hyperglycemia on macrovascular complications and cardiovascular death is a cause for concern. In this context, the regulatory agencies have modified the regulations for the approval of new drugs in diabetes, by adding the need to demonstrate that they are capable of lowering blood glucose levels together with a solid assessment of cardiovascular safety. The objective of this study is to review the cardiovascular effects of the new families of non-insulin drugs, with special emphasis on their effect on the risk of major cardiovascular events. In recent years, it has finally been confirmed that some of the drugs used to treat diabetes are not only safe from a cardiovascular point of view, but have even shown capacity to reduce the risk of cardiovascular disease in type 2 diabetes mellitus. The evidence obtained determined the updating of some current therapeutic guidelines when cardiovascular risk should be considered a fundamental variable at the time of therapeutic choice in patients with diabetes.

Cytoskeletal-like Filaments of Ca2+-Calmodulin-Dependent Protein Kinase II Are Formed in a Regulated and Zn2+-Dependent Manner.

Ca2+-calmodulin-dependent protein kinase II (CaMKII) is highly abundant in neurons, where its concentration reaches that typically found for cytoskeletal proteins. Functional reasons for such a high concentration are not known, but given the multitude of known binding partners for CaMKII, a role as a scaffolding molecule has been proposed. In this report, we provide experimental evidence that demonstrates a novel structural role for CaMKII. We discovered that CaMKII forms filaments that can extend for several micrometers in the presence of certain divalent cations (Zn2+, Cd2+, and Cu2+) but not with others (Ca2+, Mg2+, Co2+, and Ni2+). Once formed, depleting the divalent ion concentration with chelators completely dissociated the filaments, and this process could be repeated by cyclic addition and removal of divalent ions. Using the crystal structure of the CaMKII holoenzyme, we computed an electrostatic potential map of the dodecameric complex to predict divalent ion binding sites. This analysis revealed a potential surface-exposed divalent ion binding site involving amino acids that also participate in calmodulin (CaM) binding and suggested CaM binding might inhibit formation of the filaments. As predicted, Ca2+/CaM binding both inhibited divalent ion-induced filament formation and could disassemble preformed filaments. Interestingly, CaMKII within the filaments retains the capacity to autophosphorylate; however, activity toward exogenous substrates is significantly decreased. Activity is restored upon filament disassembly. We compile our results with structural and mechanistic data from the literature to propose a model of Zn2+-mediated CaMKII filament formation, in which assembly and activity are further regulated by Ca2+/CaM.

Conformational Selection and Submillisecond Dynamics of the Ligand-binding Domain of the N-Methyl-d-aspartate Receptor.

The N-methyl-d-aspartate (NMDA) receptors are heteromeric non-selective cation channels that require the binding of glycine and glutamate for gating. Based on crystal structures, the mechanism of partial agonism at the glycine-binding site is thought to be mediated by a shift in the conformational equilibrium between an open clamshell and a closed clamshell-like structure of the bilobed ligand-binding domain (LBD). Using single-molecule Förster resonance energy transfer (smFRET) and multiparameter fluorescence detection, which allows us to study the conformational states and dynamics in the submillisecond time scale, we show that there are at least three conformational states explored by the LBD: the low FRET, medium FRET, and high FRET states. The distance of the medium and low FRET states corresponds to what has been observed in crystallography structures. We show that the high FRET state, which would represent a more closed clamshell conformation than that observed in the crystal structure, is most likely the state initiating activation, as evidenced by the fact that the fraction of the protein in this state correlates well with the extent of activation. Furthermore, full agonist bound LBDs show faster dynamic motions between the medium and high FRET states, whereas they show slower dynamics when bound to weaker agonists or to antagonists.

Relative Cosolute Size Influences the Kinetics of Protein-Protein Interactions.

Protein signaling occurs in crowded intracellular environments, and while high concentrations of macromolecules are postulated to modulate protein-protein interactions, analysis of their impact at each step of the reaction pathway has not been systematically addressed. Potential cosolute-induced alterations in target association are particularly important for a signaling molecule like calmodulin (CaM), where competition among >300 targets governs which pathways are selectively activated. To explore how high concentrations of cosolutes influence CaM-target affinity and kinetics, we methodically investigated each step of the CaM-target binding mechanism under crowded or osmolyte-rich environments mimicked by ficoll-70, dextran-10, and sucrose. All cosolutes stabilized compact conformers of CaM and modulated association kinetics by affecting diffusion and rates of conformational change; however, the results showed that differently sized molecules had variable effects to enhance or impede unique steps of the association pathway. On- and off-rates were modulated by all cosolutes in a compensatory fashion, producing little change in steady-state affinity. From this work insights were gained on how high concentrations of inert crowding agents and osmolytes fit into a kinetic framework to describe protein-protein interactions relevant for cellular signaling.

Medium-term glycemic control in diabetics before coronary bypass surgery.

The aim of this study was to determine the association between preoperative medium-term (60-90 days) glycemic control, as reflected by glycosylated hemoglobin levels (HbA1c), and the incidence of major complications (mediastinitis, perioperative infarction, heart failure, stroke and kidney failure dialysis) and mortality in diabetic patients undergoing elective coronary artery by-pass graft surgery (CABG). This study suggests that aggressive glycemic control three months before surgery, achieving HbA1c=7% improvement results with less postoperative morbidity and mortality.

Time-heterogeneity of the Förster Radius from Dipole Orientational Dynamics Explains Observed Dynamic Shift.

Förster resonance energy transfer (FRET) is a quantum mechanical phenomenon involving the non-radiative transfer of energy between coupled electric dipoles. Due to the strong dependence of FRET on the distance between the dipoles, it is frequently used as a "molecular ruler" in biology, chemistry, and physics. This is done by placing dipolar molecules called dyes on molecules of interest. In time-resolved confocal single-molecule FRET (smFRET) experiments, the joint distribution of the FRET efficiency and the donor fluorescence lifetime can reveal underlying molecular conformational dynamics via deviation from their theoretical Förster relationship. This deviation is referred to as a dynamic shift. Quantifying the dynamic shift caused by the motion of the fluorescent dyes is essential to decoupling the dynamics of the studied molecules and the dyes. We develop novel Langevin models for the dye linker dynamics, including rotational dynamics, based on first physics principles and proper dye linker chemistry to match accessible volumes predicted by molecular dynamics simulations. By simulating the dyes' stochastic translational and rotational dynamics, we show that the observed dynamic shift can largely be attributed to the mutual orientational dynamics of the electric dipole moments associated with the dyes, not their accessible volume.

DNA controls the dimerization of the human FoxP1 forkhead domain.

Transcription factors (TFs) regulate gene expression by binding to specific DNA sequences and gating access to genes. Even when the binding of TFs and their cofactors to DNA is reversible, indicating a reversible control of gene expression, there is little knowledge about the molecular effect DNA has on TFs. Using single-molecule multiparameter fluorescence spectroscopy, molecular dynamics simulations, and biochemical assays, we find that the monomeric form of the forkhead (FKH) domain of the human FoxP1 behaves as a disordered protein and increases its folded population when it dimerizes. Notably, DNA binding promotes a disordered FKH dimer bound to DNA, negatively controlling the stability of the dimeric FoxP1:DNA complex. The DNA-mediated reversible regulation on FKH dimers suggests that FoxP1-dependent gene suppression is unstable, and it must require the presence of other dimerization domains or cofactors to revert the negative impact exerted by the DNA.

Coevolution and smFRET Enhances Conformation Sampling and FRET Experimental Design in Tandem PDZ1-2 Proteins.

The structural flexibility of proteins is crucial for their functions. Many experimental and computational approaches can probe protein dynamics across a range of time and length-scales. Integrative approaches synthesize the complementary outputs of these techniques and provide a comprehensive view of the dynamic conformational space of proteins, including the functionally relevant limiting conformational states and transition pathways between them. Here, we introduce an integrative paradigm to model the conformational states of multidomain proteins. As a model system, we use the first two tandem PDZ domains of postsynaptic density protein 95. First, we utilize available sequence information collected from genomic databases to identify potential amino acid interactions in the PDZ1-2 tandem that underlie modeling of the functionally relevant conformations maintained through evolution. This was accomplished through combination of coarse-grained structural modeling with outputs from direct coupling analysis measuring amino acid coevolution, a hybrid approach called SBM+DCA. We recapitulated five distinct, experimentally derived PDZ1-2 tandem conformations. In addition, SBM+DCA unveiled an unidentified, twisted conformation of the PDZ1-2 tandem. Finally, we implemented an integrative framework for the design of single-molecule Förster resonance energy transfer (smFRET) experiments incorporating the outputs of SBM+DCA with simulated FRET observables. This resulting FRET network is designed to mutually resolve the predicted limiting state conformations through global analysis. Using simulated FRET observables, we demonstrate that structural modeling with the newly designed FRET network is expected to outperform a previously used empirical FRET network at resolving all states simultaneously. Integrative approaches to experimental design have the potential to provide a new level of detail in characterizing the evolutionarily conserved conformational landscapes of proteins, and thus new insights into functional relevance of protein dynamics in biological function.

Increasing Signal Intensity of Fluorescent Oligo-Labeled Antibodies.

While full-spectrum flow cytometry has increased antibody-based multiplexing, yet further increases remain potentially impactful. We recently proposed how fluorescence Multiplexing using Spectral Imaging and Combinatorics (MuSIC) could do so using tandem dyes and an oligo-based antibody labeling method. In this work, we found that such labeled antibodies had significantly lower signal intensity than conventionally-labeled antibodies in human cell experiments. To improve signal intensity, we tested moving the fluorophores from the original external (ext.) 5' or 3' end-labeled orientation to internal (int.) fluorophore modifications. Cell-free spectrophotometer measurements showed a ~6-fold signal intensity increase of the new int. configuration compared to the previous ext. configuration. Time-resolved fluorescence spectroscopy and fluorescence correlation spectroscopy showed that ~3-fold brightness difference is due to static quenching. Spectral flow cytometry experiments using peripheral blood mononuclear cells stained with anti-CD8 antibodies showed that int. MuSIC probe-labeled antibodies have signal intensity equal to or greater than conventionally-labeled antibodies with similar estimated proportion of CD8+ lymphocytes. The antibody labeling approach is general and can be broadly applied to many biological and diagnostic applications.

DNA facilitates heterodimerization between human transcription factors FoxP1 and FoxP2 by increasing their conformational flexibility.

Transcription factors regulate gene expression by binding to DNA. They have disordered regions and specific DNA-binding domains. Binding to DNA causes structural changes, including folding and interactions with other molecules. The FoxP subfamily of transcription factors in humans is unique because they can form heterotypic interactions without DNA. However, it is unclear how they form heterodimers and how DNA binding affects their function. We used computational and experimental methods to study the structural changes in FoxP1's DNA-binding domain when it forms a heterodimer with FoxP2. We found that FoxP1 has complex and diverse conformational dynamics, transitioning between compact and extended states. Surprisingly, DNA binding increases the flexibility of FoxP1, contrary to the typical folding-upon-binding mechanism. In addition, we observed a 3-fold increase in the rate of heterodimerization after FoxP1 binds to DNA. These findings emphasize the importance of structural flexibility in promoting heterodimerization to form transcriptional complexes.

Theory for High-Throughput Genetic Interaction Screening.

Systematic, genome-scale genetic screens have been instrumental for elucidating genotype-phenotype relationships, but approaches for probing genetic interactions have been limited to at most ∼100 pre-selected gene combinations in mammalian cells. Here, we introduce a theory for high-throughput genetic interaction screens. The theory extends our recently developed Multiplexing using Spectral Imaging and Combinatorics (MuSIC) approach to propose ∼105 spectrally unique, genetically encoded MuSIC barcodes from 18 currently available fluorescent proteins. Simulation studies based on constraints imposed by spectral flow cytometry equipment suggest that genetic interaction screens at the human genome-scale may be possible if MuSIC barcodes can be paired to guide RNAs. While experimental testing of this theory awaits, it offers transformative potential for genetic perturbation technology and knowledge of genetic function. More broadly, the availability of a genome-scale spectral barcode library for non-destructive identification of single cells could find more widespread applications such as traditional genetic screening and high-dimensional lineage tracing.

Filtered FCS: species auto- and cross-correlation functions highlight binding and dynamics in biomolecules.

An analysis method of lifetime, polarization and spectrally filtered fluorescence correlation spectroscopy, referred to as filtered FCS (fFCS), is introduced. It uses, but is not limited to, multiparameter fluorescence detection to differentiate between molecular species with respect to their fluorescence lifetime, polarization and spectral information. Like the recently introduced fluorescence lifetime correlation spectroscopy (FLCS) [Chem. Phys. Lett. 2002, 353, 439-445], fFCS is based on pulsed laser excitation. However, it uses the species-specific polarization and spectrally resolved fluorescence decays to generate filters. We determined the most efficient method to generate global filters taking into account the anisotropy information. Thus, fFCS is able to distinguish species, even if they have very close or the same fluorescence lifetime, given differences in other fluorescence parameters. fFCS can be applied as a tool to compute species-specific auto- (SACF) and cross- correlation (SCCF) functions from a mixture of different species for accurate and quantitative analysis of their concentration, diffusion and kinetic properties. The computed correlation curves are also free from artifacts caused by unspecific background signal. We tested this methodology by simulating the extreme case of ligand-receptor binding processes monitored only by differences in fluorescence anisotropy. Furthermore, we apply fFCS to an experimental single-molecule FRET study of an open-to-closed conformational transition of the protein Syntaxin-1. In conclusion, fFCS and the global analysis of the SACFs and SCCF is a key tool to investigate binding processes and conformational dynamics of biomolecules in a nanosecond-to-millisecond time range as well as to unravel the involved molecular states.