PARP1-DNA co-condensation drives DNA repair site assembly to prevent disjunction of broken DNA ends.

DNA double-strand breaks (DSBs) are repaired at DSB sites. How DSB sites assemble and how broken DNA is prevented from separating is not understood. Here we uncover that the synapsis of broken DNA is mediated by the DSB sensor protein poly(ADP-ribose) (PAR) polymerase 1 (PARP1). Using bottom-up biochemistry, we reconstitute functional DSB sites and show that DSB sites form through co-condensation of PARP1 multimers with DNA. The co-condensates exert mechanical forces to keep DNA ends together and become enzymatically active for PAR synthesis. PARylation promotes release of PARP1 from DNA ends and the recruitment of effectors, such as Fused in Sarcoma, which stabilizes broken DNA ends against separation, revealing a finely orchestrated order of events that primes broken DNA for repair. We provide a comprehensive model for the hierarchical assembly of DSB condensates to explain DNA end synapsis and the recruitment of effector proteins for DNA damage repair.

Glutamate helps unmask the differences in driving forces for phase separation versus clustering of FET family proteins in sub-saturated solutions.

Multivalent proteins undergo coupled segregative and associative phase transitions. Phase separation, a segregative transition, is driven by macromolecular solubility, and this leads to coexisting phases above system-specific saturation concentrations. Percolation is a continuous transition that is driven by multivalent associations among cohesive motifs. Contributions from percolation are highlighted by the formation of heterogeneous distributions of clusters in sub-saturated solutions, as was recently reported for Fused in sarcoma (FUS) and FET family proteins. Here, we show that clustering and phase separation are defined by a separation of length- and energy-scales. This is unmasked when glutamate is the primary solution anion. Glutamate is preferentially excluded from protein sites, and this enhances molecular associations. Differences between glutamate and chloride are manifest at ultra-low protein concentrations. These differences are amplified as concentrations increase, and they saturate as the micron-scale is approached. Therefore, condensate formation in supersaturated solutions and clustering in sub-saturated are governed by distinct energy and length scales. Glutamate, unlike chloride, is the dominant intracellular anion, and the separation of scales, which is masked in chloride, is unmasked in glutamate. Our work highlights how components of cellular milieus and sequence-encoded interactions contribute to amplifying distinct contributions from associative versus segregative phase transitions.

Glutamate helps unmask the differences in driving forces for phase separation versus clustering of FET family proteins in sub-saturated solutions.

Multivalent proteins undergo coupled segregative and associative phase transitions. Phase separation, a segregative transition, is driven by macromolecular solubility, and this leads to coexisting phases above system-specific saturation concentrations. Percolation is a continuous transition that is driven by multivalent associations among cohesive motifs. Contributions from percolation are highlighted by the formation of heterogeneous distributions of clusters in sub-saturated solutions, as was recently reported for Fused in sarcoma (FUS) and FET family proteins. Here, we show that clustering and phase separation are defined by a separation of length- and energy-scales. This is unmasked when glutamate is the primary solution anion. Glutamate is preferentially excluded from protein sites, and this enhances molecular associations. Differences between glutamate and chloride are manifest at ultra-low protein concentrations. These differences are amplified as concentrations increase, and they saturate as the micron-scale is approached. Therefore, condensate formation in supersaturated solutions and clustering in sub-saturated are governed by distinct energy and length scales. Glutamate, unlike chloride, is the dominant intracellular anion, and the separation of scales, which is masked in chloride, is unmasked in glutamate. Our work highlights how components of cellular milieus and sequence-encoded interactions contribute to amplifying distinct contributions from associative versus segregative phase transitions.

A comprehensive analysis of RAS-effector interactions reveals interaction hotspots and new binding partners.

RAS effectors specifically interact with GTP-bound RAS proteins to link extracellular signals to downstream signaling pathways. These interactions rely on two types of domains, called RAS-binding (RB) and RAS association (RA) domains, which share common structural characteristics. Although the molecular nature of RAS-effector interactions is well-studied for some proteins, most of the RA/RB-domain-containing proteins remain largely uncharacterized. Here, we searched through human proteome databases, extracting 41 RA domains in 39 proteins and 16 RB domains in 14 proteins, each of which can specifically select at least one of the 25 members in the RAS family. We next comprehensively investigated the sequence-structure-function relationship between different representatives of the RAS family, including HRAS, RRAS, RALA, RAP1B, RAP2A, RHEB1, and RIT1, with all members of RA domain family proteins (RASSFs) and the RB-domain-containing CRAF. The binding affinity for RAS-effector interactions, determined using fluorescence polarization, broadly ranged between high (0.3 µM) and very low (500 µM) affinities, raising interesting questions about the consequence of these variable binding affinities in the regulation of signaling events. Sequence and structural alignments pointed to two interaction hotspots in the RA/RB domains, consisting of an average of 19 RAS-binding residues. Moreover, we found novel interactions between RRAS1, RIT1, and RALA and RASSF7, RASSF9, and RASSF1, respectively, which were systematically explored in sequence-structure-property relationship analysis, and validated by mutational analysis. These data provide a set of distinct functional properties and putative biological roles that should now be investigated in the cellular context.

Specific Conformational Dynamics and Expansion Underpin a Multi-Step Mechanism for Specific Binding of p27 with Cdk2/Cyclin A.

The protein p27, a prominent regulatory protein in eukaryotes and an intrinsically disordered protein (IDP), regulates cell division by causing cell cycle arrest when bound in ternary complex with cyclin-dependent kinase (Cdk2) and cyclins (e.g., Cdk2/Cyclin A). We present an integrative study of p27 and its binding to Cdk2/Cyclin A complex by performing single-molecule multiparameter fluorescence spectroscopy, stopped-flow experiments, and molecular dynamics simulations. Our results suggest that unbound p27 adopts a compact conformation and undergoes conformational dynamics across several orders of magnitude in time (nano-to milliseconds), reflecting a multi-step mechanism for binding Cdk2/Cyclin A. Mutagenesis studies reveal that the region D1 in p27 plays a significant role in mediating the association kinetics, undergoing conformational rearrangement upon initial binding. Additionally, FRET experiments indicate an expansion of p27 throughout binding. The detected local and long-range structural dynamics suggest that p27 exhibits a limited binding surface in the unbound form, and stochastic conformational changes in D1 facilitate initial binding to Cdk2/Cyclin A complex. Furthermore, the post-kinase inhibitory domain (post-KID) region of p27 exchanges between distinct conformational ensembles: an extended regime exhibiting worm-like chain behavior, and a compact ensemble, which may protect p27 against nonspecific interactions. In summary, the binding interaction involves three steps: (i) D1 initiates binding, (ii) p27 wraps around Cdk2/Cyclin A and D2 binds, and (iii) the fully-formed fuzzy ternary complex is formed concomitantly with an extension of the post-KID region. An understanding of how the IDP nature of p27 underpins its functional interactions with Cdk2/Cyclin A provides insight into the complex binding mechanisms of IDPs and their regulatory mechanisms.

Dynamic anticipation by Cdk2/Cyclin A-bound p27 mediates signal integration in cell cycle regulation.

p27Kip1 is an intrinsically disordered protein (IDP) that inhibits cyclin-dependent kinase (Cdk)/cyclin complexes (e.g., Cdk2/cyclin A), causing cell cycle arrest. Cell division progresses when stably Cdk2/cyclin A-bound p27 is phosphorylated on one or two structurally occluded tyrosine residues and a distal threonine residue (T187), triggering degradation of p27. Here, using an integrated biophysical approach, we show that Cdk2/cyclin A-bound p27 samples lowly-populated conformations that provide access to the non-receptor tyrosine kinases, BCR-ABL and Src, which phosphorylate Y88 or Y88 and Y74, respectively, thereby promoting intra-assembly phosphorylation (of p27) on distal T187. Even when tightly bound to Cdk2/cyclin A, intrinsic flexibility enables p27 to integrate and process signaling inputs, and generate outputs including altered Cdk2 activity, p27 stability, and, ultimately, cell cycle progression. Intrinsic dynamics within multi-component assemblies may be a general mechanism of signaling by regulatory IDPs, which can be subverted in human disease.

Real-time dynamics of peptide ligand-dependent receptor complex formation in planta.

The CLAVATA (CLV) and flagellin (flg) signaling pathways act through peptide ligands and closely related plasma membrane-localized receptor-like kinases (RLKs). The plant peptide CLV3 regulates stem cell homeostasis, whereas the bacterial flg22 peptide elicits defense responses. We applied multiparameter fluorescence imaging spectroscopy (MFIS) to characterize the dynamics of RLK complexes in the presence of ligand in living plant cells expressing receptor proteins fused to fluorescent proteins. We found that the CLV and flg pathways represent two different principles of signal transduction: flg22 first triggered RLK heterodimerization and later assembly into larger complexes through homomerization. In contrast, CLV receptor complexes were preformed, and ligand binding stimulated their clustering. This different behavior likely reflects the nature of these signaling pathways. Pathogen-triggered flg signaling impedes plant growth and development; therefore, receptor complexes are formed only in the presence of ligand. In contrast, CLV3-dependent stem cell homeostasis continuously requires active signaling, and preformation of receptor complexes may facilitate this task.

Temperature-cycle microscopy reveals single-molecule conformational heterogeneity.

Our previous temperature-cycle study reported FRET transitions between different states on FRET-labeled polyprolines [Yuan et al., PCCP, 2011, 13, 1762]. The conformational origin of such transitions, however, was left open. In this work, we apply temperature-cycle microscopy of single FRET-labeled polyproline and dsDNA molecules and compare their responses to resolve the conformational origin of different FRET states. We observe different steady-state FRET distributions and different temperature-cycle responses in the two samples. Our temperature-cycle results on single molecules resemble the results in steady-state measurements but reveal a dark state which could not be observed otherwise. By comparing the timescales and probabilities of different FRET states in temperature-cycle traces, we assign the conformational heterogeneity reflected by different FRET states to linker dynamics, dye-chain and dye-dye interactions. The dark state and low-FRET state are likely due to dye-dye interactions at short separations.

Impact of human autoantibodies on ß1-adrenergic receptor conformation, activity, and internalization.

AIMS: Autoantibodies against second extracellular loops of ß(1)-adrenergic receptors frequent in dilated cardiomyopathy confer myocardial dysfunction presumably via cAMP stimulation. Here, we investigate the autoantibody impact on receptor conformation and function. METHODS AND RESULTS: IgG was prepared from patients with dilated cardiomyopathy, matched healthy donors (10 each) or commercial IgG preparations (2). IgG binding to ß(1)-adrenergic receptor peptides was detected in 5 of 10 patients and 2 of 10 controls. IgG colocalization with the native receptor was detected in 8 of 10 patients and 1 of 10 controls (10 of 10 patients and 7 of 10 controls at >30 mg IgG/L). All IgGs exhibiting receptor colocalization triggered changes in receptor conformation (determined with fluorescent sensors) not stringently correlated to cAMP stimulation, suggesting the induction of more or less active receptor conformations. Receptor-activating IgG was detected in 8 of 10 patients but only 1 of 10 controls. In addition, IgG from 8 of 10 patients and 3 of 10 controls attenuated receptor internalization (measured by total internal reflection fluorescence microscopy). IgG-inducing inactive receptor conformations had no effect on subsequent cAMP stimulation by isoproterenol. IgG-inducing active receptor conformations dampened or augmented subsequent cAMP stimulation by isoproterenol, depending on whether receptor internalization was attenuated or not. Corresponding IgG effects on the basal beating rate and chronotropic isoproterenol response of embryonic human cardiomyocytes were observed. CONCLUSIONS: (i) Autoantibodies trigger conformation changes in the ß(1)-adrenergic receptor molecule. (ii) Some also attenuate receptor internalization. (iii) Combinations thereof increase the basal beating rate of cardiomyocytes and optionally entail dampening of their chronotropic catecholamine responses. (iv) The latter effects seem specific for patient autoantibodies, which also have higher levels.

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.

Structural heterogeneity and quantitative FRET efficiency distributions of polyprolines through a hybrid atomistic simulation and Monte Carlo approach.

Förster Resonance Energy Transfer (FRET) experiments probe molecular distances via distance dependent energy transfer from an excited donor dye to an acceptor dye. Single molecule experiments not only probe average distances, but also distance distributions or even fluctuations, and thus provide a powerful tool to study biomolecular structure and dynamics. However, the measured energy transfer efficiency depends not only on the distance between the dyes, but also on their mutual orientation, which is typically inaccessible to experiments. Thus, assumptions on the orientation distributions and averages are usually made, limiting the accuracy of the distance distributions extracted from FRET experiments. Here, we demonstrate that by combining single molecule FRET experiments with the mutual dye orientation statistics obtained from Molecular Dynamics (MD) simulations, improved estimates of distances and distributions are obtained. From the simulated time-dependent mutual orientations, FRET efficiencies are calculated and the full statistics of individual photon absorption, energy transfer, and photon emission events is obtained from subsequent Monte Carlo (MC) simulations of the FRET kinetics. All recorded emission events are collected to bursts from which efficiency distributions are calculated in close resemblance to the actual FRET experiment, taking shot noise fully into account. Using polyproline chains with attached Alexa 488 and Alexa 594 dyes as a test system, we demonstrate the feasibility of this approach by direct comparison to experimental data. We identified cis-isomers and different static local environments as sources of the experimentally observed heterogeneity. Reconstructions of distance distributions from experimental data at different levels of theory demonstrate how the respective underlying assumptions and approximations affect the obtained accuracy. Our results show that dye fluctuations obtained from MD simulations, combined with MC single photon kinetics, provide a versatile tool to improve the accuracy of distance distributions that can be extracted from measured single molecule FRET efficiencies.

The conformational dynamics of the mitochondrial Hsp70 chaperone.

Heat shock proteins 70 (Hsp70) represent a ubiquitous and conserved family of molecular chaperones involved in a plethora of cellular processes. The dynamics of their ATP hydrolysis-driven and cochaperone-regulated conformational cycle are poorly understood. We used fluorescence spectroscopy to analyze, in real time and at single-molecule resolution, the effects of nucleotides and cochaperones on the conformation of Ssc1, a mitochondrial member of the family. We report that the conformation of its ADP state is unexpectedly heterogeneous, in contrast to a uniform ATP state. Substrates are actively involved in determining the conformation of Ssc1. The J protein Mdj1 does not interact transiently with the chaperone, as generally believed, but rather is released slowly upon ATP hydrolysis. Analysis of the major bacterial Hsp70 revealed important differences between highly homologous members of the family, possibly explaining tuning of Hsp70 chaperones to meet specific functions in different organisms and cellular compartments.

Stem cell signaling in Arabidopsis requires CRN to localize CLV2 to the plasma membrane.

Stem cell number in shoot and floral meristems of Arabidopsis (Arabidopsis thaliana) is regulated by the CLAVATA3 (CLV3) signaling pathway. Perception of the CLV3 peptide requires the receptor kinase CLV1, the receptor-like protein CLV2, and the kinase CORYNE (CRN). Genetic analysis suggested that CLV2 and CRN act together and in parallel with CLV1. We studied the intracellular localization of receptor fusions with fluorescent protein tags and their capacities for interaction via efficiency of fluorescence resonance energy transfer. We found that CLV2 and CRN require each other for export from the endoplasmic reticulum and localization to the plasma membrane (PM). CRN readily forms homomers and interacts with CLV2 through the transmembrane domain and adjacent juxtamembrane sequences. CLV1 forms homomers independently of CLV2 and CRN at the PM. We propose that the CLV3 signal is perceived by a tetrameric CLV2/CRN complex and a CLV1 homodimer that localize to the PM and can interact via CRN.

Multiparameter fluorescence image spectroscopy to study molecular interactions.

Multiparameter Fluorescence Image Spectroscopy (MFIS) is used to monitor simultaneously a variety of fluorescence parameters in confocal fluorescence microscopy. As the photons are registered one by one, MFIS allows for fully parallel recording of Fluorescence Correlation/Cross Correlation Spectroscopy (FCS/FCCS), fluorescence lifetime and pixel/image information over time periods of hours with picosecond accuracy. The analysis of the pixel fluorescence information in higher-dimensional histograms maximizes the selectivity of fluorescence microscopic methods. Moreover it facilitates a statistically-relevant data analysis of the pixel information which makes an efficient detection of heterogeneities possible. The reliability of MFIS has been demonstrated for molecular interaction studies in different complex environments: (I) detecting the heterogeneity of diffusion properties of the dye Rhodamine 110 in a sepharose bead, (II) Förster Resonance Energy Transfer (FRET) studies in mammalian HEK293 cells, and (III) FRET study of the homodimerisation of the transcription factor BIM1 in plant cells. The multidimensional analysis of correlated changes of several parameters measured by FRET, FCS, fluorescence lifetime and anisotropy increases the robustness of the analysis significantly. The economic use of photon information allows one to keep the expression levels of fluorescent protein-fusion proteins as low as possible (down to the single-molecule level).

Single-molecule FRET measures bends and kinks in DNA.

We present advances in the use of single-molecule FRET measurements with flexibly linked dyes to derive full 3D structures of DNA constructs based on absolute distances. The resolution obtained by this single-molecule approach harbours the potential to study in detail also protein- or damage-induced DNA bending. If one is to generate a geometric structural model, distances between fixed positions are needed. These are usually not experimentally accessible because of unknown fluorophore-linker mobility effects that lead to a distribution of FRET efficiencies and distances. To solve this problem, we performed studies on DNA double-helices by systematically varying donor acceptor distances from 2 to 10 nm. Analysis of dye-dye quenching and fluorescence anisotropy measurements reveal slow positional and fast orientational fluorophore dynamics, that results in an isotropic average of the FRET efficiency. We use a nonlinear conversion function based on MD simulations that allows us to include this effect in the calculation of absolute FRET distances. To obtain unique structures, we performed a quantitative statistical analysis for the conformational search in full space based on triangulation, which uses the known helical nucleic acid features. Our higher accuracy allowed the detection of sequence-dependent DNA bending by 16 degrees . For DNA with bulged adenosines, we also quantified the kink angles introduced by the insertion of 1, 3 and 5 bases to be 32 degrees +/- 6 degrees , 56 degrees +/- 4 degrees and 73 +/- 2 degrees , respectively. Moreover, the rotation angles and shifts of the helices were calculated to describe the relative orientation of the two arms in detail.

Monitoring dynamic systems with multiparameter fluorescence imaging.

A new general strategy based on the use of multiparameter fluorescence detection (MFD) to register and quantitatively analyse fluorescence images is introduced. Multiparameter fluorescence imaging (MFDi) uses pulsed excitation, time-correlated single-photon counting and a special pixel clock to simultaneously monitor the changes in the eight-dimensional fluorescence information (fundamental anisotropy, fluorescence lifetime, fluorescence intensity, time, excitation spectrum, fluorescence spectrum, fluorescence quantum yield, distance between fluorophores) in real time. The three spatial coordinates are also stored. The most statistically efficient techniques known from single-molecule spectroscopy are used to estimate fluorescence parameters of interest for all pixels, not just for the regions of interest. Their statistical significance is judged from a stack of two-dimensional histograms. In this way, specific pixels can be selected for subsequent pixel-based subensemble analysis in order to improve the statistical accuracy of the parameters estimated. MFDi avoids the need for sequential measurements, because the registered data allow one to perform many analysis techniques, such as fluorescence-intensity distribution analysis (FIDA) and fluorescence correlation spectroscopy (FCS), in an off-line mode. The limitations of FCS for counting molecules and monitoring dynamics are discussed. To demonstrate the ability of our technique, we analysed two systems: (i) interactions of the fluorescent dye Rhodamine 110 inside and outside of a glutathione sepharose bead, and (ii) microtubule dynamics in live yeast cells of Schizosaccharomyces pombe using a fusion protein of Green Fluorescent Protein (GFP) with Minichromosome Altered Loss Protein 3 (Mal3), which is involved in the dynamic cycle of polymerising and depolymerising microtubules.

Proton-powered subunit rotation in single membrane-bound F0F1-ATP synthase.

Synthesis of ATP from ADP and phosphate, catalyzed by F(0)F(1)-ATP synthases, is the most abundant physiological reaction in almost any cell. F(0)F(1)-ATP synthases are membrane-bound enzymes that use the energy derived from an electrochemical proton gradient for ATP formation. We incorporated double-labeled F(0)F(1)-ATP synthases from Escherichia coli into liposomes and measured single-molecule fluorescence resonance energy transfer (FRET) during ATP synthesis and hydrolysis. The gamma subunit rotates stepwise during proton transport-powered ATP synthesis, showing three distinct distances to the b subunits in repeating sequences. The average durations of these steps correspond to catalytic turnover times upon ATP synthesis as well as ATP hydrolysis. The direction of rotation during ATP synthesis is opposite to that of ATP hydrolysis.