GPCR-mediated ß-arrestin activation deconvoluted with single-molecule precision.

ß-arrestins bind G protein-coupled receptors to terminate G protein signaling and to facilitate other downstream signaling pathways. Using single-molecule fluorescence resonance energy transfer imaging, we show that ß-arrestin is strongly autoinhibited in its basal state. Its engagement with a phosphopeptide mimicking phosphorylated receptor tail efficiently releases the ß-arrestin tail from its N domain to assume distinct conformations. Unexpectedly, we find that ß-arrestin binding to phosphorylated receptor, with a phosphorylation barcode identical to the isolated phosphopeptide, is highly inefficient and that agonist-promoted receptor activation is required for ß-arrestin activation, consistent with the release of a sequestered receptor C tail. These findings, together with focused cellular investigations, reveal that agonism and receptor C-tail release are specific determinants of the rate and efficiency of ß-arrestin activation by phosphorylated receptor. We infer that receptor phosphorylation patterns, in combination with receptor agonism, synergistically establish the strength and specificity with which diverse, downstream ß-arrestin-mediated events are directed.

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.

Driving forces of the complex formation between highly charged disordered proteins.

Highly disordered complexes between oppositely charged intrinsically disordered proteins present a new paradigm of biomolecular interactions. Here, we investigate the driving forces of such interactions for the example of the highly positively charged linker histone H1 and its highly negatively charged chaperone, prothymosin α (ProTα). Temperature-dependent single-molecule Förster resonance energy transfer (FRET) experiments and isothermal titration calorimetry reveal ProTα-H1 binding to be enthalpically unfavorable, and salt-dependent affinity measurements suggest counterion release entropy to be an important thermodynamic driving force. Using single-molecule FRET, we also identify ternary complexes between ProTα and H1 in addition to the heterodimer at equilibrium and show how they contribute to the thermodynamics observed in ensemble experiments. Finally, we explain the observed thermodynamics quantitatively with a mean-field polyelectrolyte theory that treats counterion release explicitly. ProTα-H1 complex formation resembles the interactions between synthetic polyelectrolytes, and the underlying principles are likely to be of broad relevance for interactions between charged biomolecules in general.

Extreme disorder in an ultrahigh-affinity protein complex.

Molecular communication in biology is mediated by protein interactions. According to the current paradigm, the specificity and affinity required for these interactions are encoded in the precise complementarity of binding interfaces. Even proteins that are disordered under physiological conditions or that contain large unstructured regions commonly interact with well-structured binding sites on other biomolecules. Here we demonstrate the existence of an unexpected interaction mechanism: the two intrinsically disordered human proteins histone H1 and its nuclear chaperone prothymosin-α associate in a complex with picomolar affinity, but fully retain their structural disorder, long-range flexibility and highly dynamic character. On the basis of closely integrated experiments and molecular simulations, we show that the interaction can be explained by the large opposite net charge of the two proteins, without requiring defined binding sites or interactions between specific individual residues. Proteome-wide sequence analysis suggests that this interaction mechanism may be abundant in eukaryotes.

Tuning the Baird aromatic triplet-state energy of cyclooctatetraene to maximize the self-healing mechanism in organic fluorophores.

Bright, photostable, and nontoxic fluorescent contrast agents are critical for biological imaging. "Self-healing" dyes, in which triplet states are intramolecularly quenched, enable fluorescence imaging by increasing fluorophore brightness and longevity, while simultaneously reducing the generation of reactive oxygen species that promote phototoxicity. Here, we systematically examine the self-healing mechanism in cyanine-class organic fluorophores spanning the visible spectrum. We show that the Baird aromatic triplet-state energy of cyclooctatetraene can be physically altered to achieve order of magnitude enhancements in fluorophore brightness and signal-to-noise ratio in both the presence and absence of oxygen. We leverage these advances to achieve direct measurements of large-scale conformational dynamics within single molecules at submillisecond resolution using wide-field illumination and camera-based detection methods. These findings demonstrate the capacity to image functionally relevant conformational processes in biological systems in the kilohertz regime at physiological oxygen concentrations and shed important light on the multivariate parameters critical to self-healing organic fluorophore design.

Inferring properties of disordered chains from FRET transfer efficiencies.

Förster resonance energy transfer (FRET) is a powerful tool for elucidating both structural and dynamic properties of unfolded or disordered biomolecules, especially in single-molecule experiments. However, the key observables, namely, the mean transfer efficiency and fluorescence lifetimes of the donor and acceptor chromophores, are averaged over a broad distribution of donor-acceptor distances. The inferred average properties of the ensemble therefore depend on the form of the model distribution chosen to describe the distance, as has been widely recognized. In addition, while the distribution for one type of polymer model may be appropriate for a chain under a given set of physico-chemical conditions, it may not be suitable for the same chain in a different environment so that even an apparently consistent application of the same model over all conditions may distort the apparent changes in chain dimensions with variation of temperature or solution composition. Here, we present an alternative and straightforward approach to determining ensemble properties from FRET data, in which the polymer scaling exponent is allowed to vary with solution conditions. In its simplest form, it requires either the mean FRET efficiency or fluorescence lifetime information. In order to test the accuracy of the method, we have utilized both synthetic FRET data from implicit and explicit solvent simulations for 30 different protein sequences, and experimental single-molecule FRET data for an intrinsically disordered and a denatured protein. In all cases, we find that the inferred radii of gyration are within 10% of the true values, thus providing higher accuracy than simpler polymer models. In addition, the scaling exponents obtained by our procedure are in good agreement with those determined directly from the molecular ensemble. Our approach can in principle be generalized to treating other ensemble-averaged functions of intramolecular distances from experimental data.

Single-molecule fluorescence reveals sequence-specific misfolding in multidomain proteins.

A large range of debilitating medical conditions is linked to protein misfolding, which may compete with productive folding particularly in proteins containing multiple domains. Seventy-five per cent of the eukaryotic proteome consists of multidomain proteins, yet it is not understood how interdomain misfolding is avoided. It has been proposed that maintaining low sequence identity between covalently linked domains is a mechanism to avoid misfolding. Here we use single-molecule Förster resonance energy transfer to detect and quantify rare misfolding events in tandem immunoglobulin domains from the I band of titin under native conditions. About 5.5 per cent of molecules with identical domains misfold during refolding in vitro and form an unexpectedly stable state with an unfolding half-time of several days. Tandem arrays of immunoglobulin-like domains in humans show significantly lower sequence identity between neighbouring domains than between non-adjacent domains. In particular, the sequence identity of neighbouring domains has been found to be preferentially below 40 per cent. We observe no misfolding for a tandem of naturally neighbouring domains with low sequence identity (24 per cent), whereas misfolding occurs between domains that are 42 per cent identical. Coarse-grained molecular simulations predict the formation of domain-swapped structures that are in excellent agreement with the observed transfer efficiency of the misfolded species. We infer that the interactions underlying misfolding are very specific and result in a sequence-specific domain-swapping mechanism. Diversifying the sequence between neighbouring domains seems to be a successful evolutionary strategy to avoid misfolding in multidomain proteins.

Probing the Action of Chemical Denaturant on an Intrinsically Disordered Protein by Simulation and Experiment.

Chemical denaturants are the most commonly used agents for unfolding proteins and are thought to act by better solvating the unfolded state. Improved solvation is expected to lead to an expansion of unfolded chains with increasing denaturant concentration, providing a sensitive probe of the denaturant action. However, experiments have so far yielded qualitatively different results concerning the effects of chemical denaturation. Studies using Förster resonance energy transfer (FRET) and other methods found an increase in radius of gyration with denaturant concentration, but most small-angle X-ray scattering (SAXS) studies found no change. This discrepancy therefore challenges our understanding of denaturation mechanism and more generally the accuracy of these experiments as applied to unfolded or disordered proteins. Here, we use all-atom molecular simulations to investigate the effect of urea and guanidinium chloride on the structure of the intrinsically disordered protein ACTR, which can be studied by experiment over a wide range of denaturant concentration. Using unbiased molecular simulations with a carefully calibrated denaturant model, we find that the protein chain indeed swells with increasing denaturant concentration. This is due to the favorable association of urea or guanidinium chloride with the backbone of all residues and with the side-chains of almost all residues, with denaturant-water transfer free energies inferred from this association in reasonable accord with experimental estimates. Interactions of the denaturants with the backbone are dominated by hydrogen bonding, while interactions with side-chains include other contributions. By computing FRET efficiencies and SAXS intensities at each denaturant concentration, we show that the simulation trajectories are in accord with both experiments on this protein, demonstrating that there is no fundamental inconsistency between the two types of experiment. Agreement with experiment also supports the picture of chemical denaturation described in our simulations, driven by weak association of denaturant with the protein. Our simulations support some assumptions needed for each experiment to accurately reflect changes in protein size, namely, that the commonly used FRET chromophores do not qualitatively alter the results and that possible effects such as preferential solvent partitioning into the interior of the chain do not interfere with the determination of radius of gyration from the SAXS experiments.

Consistent View of Polypeptide Chain Expansion in Chemical Denaturants from Multiple Experimental Methods.

There has been a long-standing controversy regarding the effect of chemical denaturants on the dimensions of unfolded and intrinsically disordered proteins: A wide range of experimental techniques suggest that polypeptide chains expand with increasing denaturant concentration, but several studies using small-angle X-ray scattering (SAXS) have reported no such increase of the radius of gyration (Rg). This inconsistency challenges our current understanding of the mechanism of chemical denaturants, which are widely employed to investigate protein folding and stability. Here, we use a combination of single-molecule Förster resonance energy transfer (FRET), SAXS, dynamic light scattering (DLS), and two-focus fluorescence correlation spectroscopy (2f-FCS) to characterize the denaturant dependence of the unfolded state of the spectrin domain R17 and the intrinsically disordered protein ACTR in two different denaturants. Standard analysis of the primary data clearly indicates an expansion of the unfolded state with increasing denaturant concentration irrespective of the protein, denaturant, or experimental method used. This is the first case in which SAXS and FRET have yielded even qualitatively consistent results regarding expansion in denaturant when applied to the same proteins. To more directly illustrate this self-consistency, we used both SAXS and FRET data in a Bayesian procedure to refine structural ensembles representative of the observed unfolded state. This analysis demonstrates that both of these experimental probes are compatible with a common ensemble of protein configurations for each denaturant concentration. Furthermore, the resulting ensembles reproduce the trend of increasing hydrodynamic radius with denaturant concentration obtained by 2f-FCS and DLS. We were thus able to reconcile the results from all four experimental techniques quantitatively, to obtain a comprehensive structural picture of denaturant-induced unfolded state expansion, and to identify the most likely sources of earlier discrepancies.

Experimental evidence for a frustrated energy landscape in a three-helix-bundle protein family.

Energy landscape theory is a powerful tool for understanding the structure and dynamics of complex molecular systems, in particular biological macromolecules. The primary sequence of a protein defines its free-energy landscape and thus determines the folding pathway and the rate constants of folding and unfolding, as well as the protein's native structure. Theory has shown that roughness in the energy landscape will lead to slower folding, but derivation of detailed experimental descriptions of this landscape is challenging. Simple folding models show that folding is significantly influenced by chain entropy; proteins in which the contacts are local fold quickly, owing to the low entropy cost of forming stabilizing, native contacts during folding. For some protein families, stability is also a determinant of folding rate constants. Where these simple metrics fail to predict folding behaviour, it is probable that there are features in the energy landscape that are unusual. Such general observations cannot explain the folding behaviour of the R15, R16 and R17 domains of alpha-spectrin. R15 folds approximately 3,000 times faster than its homologues, although they have similar structures, stabilities and, as far as can be determined, transition-state stabilities. Here we show that landscape roughness (internal friction) is responsible for the slower folding and unfolding of R16 and R17. We use chimaeric domains to demonstrate that this internal friction is a property of the core, and suggest that frustration in the landscape of the slow-folding spectrin domains may be due to misdocking of the long helices during folding. Theoretical studies have suggested that rugged landscapes will result in slower folding; here we show experimentally that such a phenomenon directly influences the folding kinetics of a 'normal' protein, that is, one with a significant energy barrier that folds on a relatively slow, millisecond-second, timescale.

Polymer scaling laws of unfolded and intrinsically disordered proteins quantified with single-molecule spectroscopy.

The dimensions of unfolded and intrinsically disordered proteins are highly dependent on their amino acid composition and solution conditions, especially salt and denaturant concentration. However, the quantitative implications of this behavior have remained unclear, largely because the effective theta-state, the central reference point for the underlying polymer collapse transition, has eluded experimental determination. Here, we used single-molecule fluorescence spectroscopy and two-focus correlation spectroscopy to determine the theta points for six different proteins. While the scaling exponents of all proteins converge to 0.62 ± 0.03 at high denaturant concentrations, as expected for a polymer in good solvent, the scaling regime in water strongly depends on sequence composition. The resulting average scaling exponent of 0.46 ± 0.05 for the four foldable protein sequences in our study suggests that the aqueous cellular milieu is close to effective theta conditions for unfolded proteins. In contrast, two intrinsically disordered proteins do not reach the Θ-point under any of our solvent conditions, which may reflect the optimization of their expanded state for the interactions with cellular partners. Sequence analyses based on our results imply that foldable sequences with more compact unfolded states are a more recent result of protein evolution.

Release of linker histone from the nucleosome driven by polyelectrolyte competition with a disordered protein.

Highly charged intrinsically disordered proteins are essential regulators of chromatin structure and transcriptional activity. Here we identify a surprising mechanism of molecular competition that relies on the pronounced dynamical disorder present in these polyelectrolytes and their complexes. The highly positively charged human linker histone H1.0 (H1) binds to nucleosomes with ultrahigh affinity, implying residence times incompatible with efficient biological regulation. However, we show that the disordered regions of H1 retain their large-amplitude dynamics when bound to the nucleosome, which enables the highly negatively charged and disordered histone chaperone prothymosin α to efficiently invade the H1-nucleosome complex and displace H1 via a competitive substitution mechanism, vastly accelerating H1 dissociation. By integrating experiments and simulations, we establish a molecular model that rationalizes the remarkable kinetics of this process structurally and dynamically. Given the abundance of polyelectrolyte sequences in the nuclear proteome, this mechanism is likely to be widespread in cellular regulation.

Quantifying heterogeneity and conformational dynamics from single molecule FRET of diffusing molecules: recurrence analysis of single particles (RASP).

Single molecule Förster resonance energy transfer (FRET) experiments are a versatile method for investigating the conformational distributions and dynamics of biological macromolecules. In a common type of experiment, the fluorescence bursts from individual molecules freely diffusing in solution are detected as they pass through the observation volume of a confocal microscope. Correlation analysis of the fluorescence bursts shows that under typical experimental conditions, for time scales up to several tens of milliseconds, the probability for a molecule to return to the confocal volume is greater than the probability of a new molecule being detected. Here we present RASP (recurrence analysis of single particles), a method that is based on this recurrence behavior and allows us to significantly extend the information that can be extracted from single molecule FRET experiments. The number and peak shapes of subpopulations within the sample can be identified essentially in a model-free way by constructing recurrence FRET efficiency histograms. These are obtained by first selecting photon bursts from a small transfer efficiency range (initial bursts), and then building the FRET efficiency histogram only from bursts detected within a short time (the recurrence interval) after the initial bursts. Systematic variation of the recurrence interval allows the kinetics of interconversion between subpopulations to be determined on time scales from ~50 µs up to ~100 ms from equilibrium measurements. We demonstrate the applicability of the method on measurements of several peptides and proteins with different degrees of conformational heterogeneity and folding dynamics. The concepts presented here can be extended to other observables available from single molecule fluorescence experiments.

Binding without folding - the biomolecular function of disordered polyelectrolyte complexes.

Recent evidence shows that oppositely charged intrinsically disordered proteins (IDPs) can form high-affinity complexes that involve neither the formation of secondary or tertiary structure nor site-specific interactions between individual residues. Similar electrostatically dominated interactions have also been identified for positively charged IDPs binding to nucleic acids. These highly disordered polyelectrolyte complexes constitute an extreme case within the spectrum of biomolecular interactions involving disorder. Such interactions are likely to be widespread, since sequence analysis predicts proteins with highly charged disordered regions to be surprisingly numerous. Here, we summarize the insights that have emerged from the highly disordered polyelectrolyte complexes identified so far, and we highlight recent developments and future challenges in (i) establishing models for the underlying highly dynamic structural ensembles, (ii) understanding the novel binding mechanisms associated with them, and (iii) identifying the functional consequences.

Polyelectrolyte interactions enable rapid association and dissociation in high-affinity disordered protein complexes.

Highly charged intrinsically disordered proteins can form complexes with very high affinity in which both binding partners fully retain their disorder and dynamics, exemplified by the positively charged linker histone H1.0 and its chaperone, the negatively charged prothymosin α. Their interaction exhibits another surprising feature: The association/dissociation kinetics switch from slow two-state-like exchange at low protein concentrations to fast exchange at higher, physiologically relevant concentrations. Here we show that this change in mechanism can be explained by the formation of transient ternary complexes favored at high protein concentrations that accelerate the exchange between bound and unbound populations by orders of magnitude. Molecular simulations show how the extreme disorder in such polyelectrolyte complexes facilitates (i) diffusion-limited binding, (ii) transient ternary complex formation, and (iii) fast exchange of monomers by competitive substitution, which together enable rapid kinetics. Biological polyelectrolytes thus have the potential to keep regulatory networks highly responsive even for interactions with extremely high affinities.

Fast folding kinetics and stabilization of apo-cytochrome c.

It is generally accepted that in the c-type cytochromes the covalently bound heme plays a primary role in the acquisition of the folded state. Here, we show that a stabilized site-directed variant of apo-cyt c551 from Pseudomonas aeruginosa (Pa-apocyt F7A/W77F) retains native-like features in the presence of sodium sulfate even in the absence of heme. By time-resolved intrinsic fluorescence, we have evidence that Pa-apocyt F7A/W77F may acquire a compact, native-like conformation within microseconds. These results challenge current thinking about the role of the heme group in the folding of c-type cytochromes.

Cross-View Discriminative Feature Learning for Person Re-Identification.

The viewpoint variability across a network of non-overlapping cameras is a challenging problem affecting person re-identification performance. In this paper, we investigate how to mitigate the cross-view ambiguity by learning highly discriminative deep features under the supervision of a novel loss function. The proposed objective is made up of two terms, the steering meta center term and the enhancing centers dispersion term, that steer the training process to mining effective intra-class and inter-class relationships in the feature domain of the identities. The effect of our loss supervision is to generate a more expanded feature space of compact classes where the overall level of the inter-identities' interference is reduced. Compared with the existing metric learning techniques, this approach has the advantage of achieving a better optimization because it jointly learns the embedding and the metric contextually. Our technique, by dismissing side-sources of performance gain, proves to enhance the CNN invariance to viewpoint without incurring increased training complexity (like in Siamese or triplet networks) and outperforms many related state-of-the-art techniques on Market-1501 and CUHK03.

Comment on "Innovative scattering analysis shows that hydrophobic disordered proteins are expanded in water".

Riback et al (Reports, 13 October 2017, p. 238) used small-angle x-ray scattering (SAXS) experiments to infer a degree of compaction for unfolded proteins in water versus chemical denaturant that is highly consistent with the results from Förster resonance energy transfer (FRET) experiments. There is thus no "contradiction" between the two methods, nor evidence to support their claim that commonly used FRET fluorophores cause protein compaction.

Empirical Optimization of Interactions between Proteins and Chemical Denaturants in Molecular Simulations.

Chemical denaturants are the most commonly used perturbation applied to study protein stability and folding kinetics as well as the properties of unfolded polypeptides. We build on recent work balancing the interactions of proteins and water, and accurate models for the solution properties of urea and guanidinium chloride, to develop a combined force field that is able to capture the strength of interactions between proteins and denaturants. We use solubility data for a model tetraglycine peptide in each denaturant to tune the protein-denaturant interaction by a novel simulation methodology. We validate the results against data for more complex sequences: single-molecule Förster resonance energy transfer data for a 34-residue fragment of the globular protein CspTm and photoinduced electron transfer quenching data for the disordered peptides C(AGQ)nW in denaturant solution as well as the chemical denaturation of the mini-protein Trp cage. The combined force field model should aid our understanding of denaturation mechanisms and the interpretation of experiment.