Photon bursts from single diffusing donoracceptor labeled macromolecules were used to measure intramolecular distances and identify subpopulations of freely diffusing macromolecules in a heterogeneous ensemble. By using DNA as a rigid spacer, a series of constructs with varying intramolecular donor-acceptor spacings were used to measure the mean and distribution width of f luorescence resonance energy transfer (FRET) efficiencies as a function of distance. The mean single-pair FRET efficiencies qualitatively follow the distance dependence predicted by Förster theory. Possible contributions to the widths of the FRET efficiency distributions are discussed, and potential applications in the study of biopolymer conformational dynamics are suggested. The ability to measure intramolecular (and intermolecular) distances for single molecules implies the ability to distinguish and monitor subpopulations of molecules in a mixture with different distances or conformational states. This is demonstrated by monitoring substrate and product subpopulations before and after a restriction endonuclease cleavage reaction. Distance measurements at single-molecule resolution also should facilitate the study of complex reactions such as biopolymer folding. To this end, the denaturation of a DNA hairpin was examined by using single-pair FRET.During the past decade, spectacular progress has been made in the field of single-molecule fluorescence detection (1-3). Single-molecule experiments enable us to study physical, chemical, or biological properties of molecules on a truly molecular basis, in contrast to conventional methods, which only provide ensemble averaged data. Thus, single-molecule experiments allow a direct comparison to theory, which usually analyzes systems at the microscopic level.Single fluorescently tagged biomolecules can be detected when they are freely diffusing or flowing in solutions or when they are immobilized (surface attached or imbedded in matrices or droplets), providing different and complementary kinds of information. With immobilized molecules, the evolution of a property for a particular molecule over an extended period of time can be studied. This allows, for example, the observation of its dynamic fluctuations under equilibrium conditions, or acquisition of its full time-trajectory during a reaction (4). However, care must be taken to ensure minimal perturbation from the immobilization.When diffusing or flowing single molecules in a liquid traverse the laser excitation volume, fluorescence photon bursts are generated. Such bursts can be analyzed for their brightness, duration, spectrum, and fluorescence lifetime, thereby providing molecular information on identity, size, diffusion coefficient, and concentration (5-7). These bursts are short (millisecond) and provide less information on slower conformational dynamics. However, they can provide invaluable information about the distributions of molecular properties of interest, undisturbed by surface effects. Because a large number of events (photon...

The nucleolus is a membrane-less organelle formed through liquid-liquid phase separation of its components from the surrounding nucleoplasm. Here, we show that nucleophosmin (NPM1) integrates within the nucleolus via a multi-modal mechanism involving multivalent interactions with proteins containing arginine-rich linear motifs (R-motifs) and ribosomal RNA (rRNA). Importantly, these R-motifs are found in canonical nucleolar localization signals. Based on a novel combination of biophysical approaches, we propose a model for the molecular organization within liquid-like droplets formed by the N-terminal domain of NPM1 and R-motif peptides, thus providing insights into the structural organization of the nucleolus. We identify multivalency of acidic tracts and folded nucleic acid binding domains, mediated by N-terminal domain oligomerization, as structural features required for phase separation of NPM1 with other nucleolar components in vitro and for localization within mammalian nucleoli. We propose that one mechanism of nucleolar localization involves phase separation of proteins within the nucleolus.DOI: http://dx.doi.org/10.7554/eLife.13571.001

We studied the coupled binding and folding of ␣-synuclein, an intrinsically disordered protein linked with Parkinson's disease. Using single-molecule fluorescence resonance energy transfer and correlation methods, we directly probed protein membrane association, structural distributions, and dynamics. Results revealed an intricate energy landscape on which binding of ␣-synuclein to amphiphilic small molecules or membrane-like partners modulates conformational transitions between a natively unfolded state and multiple ␣-helical structures. ␣-Synuclein conformation is not continuously tunable, but instead partitions into 2 main classes of folding landscape structural minima. The switch between a broken and an extended helical structure can be triggered by changing the concentration of binding partners or by varying the curvature of the binding surfaces presented by micelles or bilayers composed of the lipid-mimetic SDS. Single-molecule experiments with lipid vesicles of various composition showed that a low fraction of negatively charged lipids, similar to that found in biological membranes, was sufficient to drive ␣-synuclein binding and folding, resulting here in the induction of an extended helical structure. Overall, our results imply that the 2 folded structures are preencoded by the ␣-synuclein amino acid sequence, and are tunable by small-molecule supramolecular states and differing membrane properties, suggesting novel control elements for biological and amyloid regulation of ␣-synuclein.A lpha-synuclein, a highly acidic 140-residue protein expressed at high levels in the human brain and enriched in presynaptic nerve termini, is a member of the growing class of intrinsically disordered proteins that adopt ordered structure upon interaction with cellular partners (1-5). This natively unfolded protein plays crucial roles in the pathogenesis of several neurodegenerative disorders including Parkinson's disease and Alzheimer's disease (6-9). Although several physiological functions have been proposed for the protein, including roles in the regulation of distinct pools of presynaptic vesicles (10, 11), maintenance of SNARE protein complexes (12), modulation of neural plasticity (13), control of dopamine neurotransmission (14), and ER-Golgi trafficking (15), its precise biological role remains unclear. Nevertheless, membrane interaction is generally believed to be a key modulator of ␣-synuclein function (16,17).Sequence analysis predicts ␣-synuclein interaction with lipid membranes through amphipathic ␣-helices encoded by 7 imperfect 11-residue repeats, approximately 4 of which are located in the highly basic N-terminal region of the protein, and 3 in the highly acidic and hydrophobic NAC region (non-A␤ component of Alzheimer's disease amyloid) (13, 18). Not surprisingly, the protein undergoes structural transitions upon binding to either brain-derived or synthetic acidic phospholipid vesicles, adopting ␣-helical conformations in the membrane-bound form (17)(18)(19). Similarly, ␣-synuclein assumes helical structu...

Fluorescence resonance energy transfer and f luorescence polarization anisotropy are used to investigate single molecules of the enzyme staphylococcal nuclease. Intramolecular f luorescence resonance energy transfer and f luorescence polarization anisotropy measurements of f luorescently labeled staphylococcal nuclease molecules reveal distinct patterns of f luctuations that may be attributed to protein conformational dynamics on the millisecond time scale. Intermolecular f luorescence resonance energy transfer measurements provide information about the dynamic interactions of staphylococcal nuclease with single substrate molecules. The experimental methods demonstrated here should prove generally useful in studies of protein folding and enzyme catalysis at single-molecule resolution.Single-molecule spectroscopy can provide information about complex biological molecules and systems that is difficult to obtain from ensemble measurements (1-6). For example, one can observe the time trajectories of single molecules in biochemical reactions that cannot be synchronized in ensemble experiments. In a population of molecules heterogeneous in a particular property, single-molecule spectroscopy can also resolve and quantitatively compare distinct subpopulations that would be indistinguishable at the ensemble level.Fluorescence resonance energy transfer (FRET) measurements between single pairs of acceptor and donor fluorophores can yield information about structural relationships and distance fluctuations between regions of a single biomolecule or between components of an interacting system of biomolecules (7-10). In addition, the rotational dynamics of a single fluorophore can be probed by monitoring fluctuations in fluorescence polarization (11)(12)(13)(14). Here we develop the techniques of single-pair FRET (spFRET) and singlemolecule fluorescence polarization anisotropy (smFPA) and show how they can be used to observe the conformational fluctuations and catalytic reactions of enzymes at singlemolecule resolution.Staphylococcal nuclease (SNase) is a 19 kDa Ca 2ϩ -dependent enzyme that catalyzes the hydrolysis of DNA and RNA into mono-and dinucleotides (15). Its catalytic mechanism, thermodynamic stability, and folding pathway have been studied extensively at the ensemble level (16-21). To probe the conformational dynamics of SNase and its interactions with substrate at single-molecule resolution, three experimental methods were used. First, intramolecular spFRET was measured between donor and acceptor fluorophores attached to single SNase proteins. Second, single-molecule fluorescence polarization anisotropy measurements were performed by using SNase labeled singly with one type of fluorophore. Third, intermolecular spFRET was measured between donor-labeled SNase and acceptor-labeled DNA substrate.Using intramolecular spFRET measurements on single SNase protein molecules, we observe interesting dynamics including gradual fluctuations in the FRET efficiencies. A combination of smFPA measurements, simulations, and...

We report single-molecule folding studies of a small, single-domain protein, chymotrypsin inhibitor 2 (CI2). CI2 is an excellent model system for protein folding studies and has been extensively studied, both experimentally (at the ensemble level) and theoretically. Conformationally assisted ligation methodology was used to synthesize the proteins and site-specifically label them with donor and acceptor dyes. Folded and denatured subpopulations were observed by fluorescence resonance energy transfer (FRET) measurements on freely diffusing single protein molecules. Properties of these subpopulations were directly monitored as a function of guanidinium chloride concentration. It is shown that new information about different aspects of the protein folding reaction can be extracted from such subpopulation properties. Shifts in the mean transfer efficiencies are discussed, FRET efficiency distributions are translated into potentials, and denaturation curves are directly plotted from the areas of the FRET peaks. Changes in stability caused by mutation also are measured by comparing pseudo wild-type CI2 with a destabilized mutant (K17G). Current limitations and future possibilities and prospects for single-pair FRET protein folding investigations are discussed.

Nucleophosmin (NPM1) is an abundant, oligomeric protein in the granular component of the nucleolus with roles in ribosome biogenesis. Pentameric NPM1 undergoes liquid-liquid phase separation (LLPS) via heterotypic interactions with nucleolar components, including ribosomal RNA (rRNA) and proteins which display multivalent arginine-rich linear motifs (Rmotifs), and is integral to the liquid-like nucleolar matrix. Here we show that NPM1 can also undergo LLPS via homotypic interactions between its polyampholytic intrinsically disordered regions, a mechanism that opposes LLPS via heterotypic interactions. Using a combination of biophysical techniques, including confocal microscopy, SAXS, analytical ultracentrifugation, and single-molecule fluorescence, we describe how conformational changes within NPM1 control valency and switching between the different LLPS mechanisms. We propose that this newly discovered interplay between multiple LLPS mechanisms may influence the direction of vectorial pre-ribosomal particle assembly within, and exit from the nucleolus as part of the ribosome biogenesis process.

Intracellular ribonucleoprotein (RNP) granules are membrane-less droplet organelles that are thought to regulate posttranscriptional gene expression. While liquid-liquid phase separation may drive RNP granule assembly, the mechanisms underlying their supramolecular dynamics and internal organization remain poorly understood. Here we demonstrate that RNA, a primary component of RNP granules, can modulate the phase behavior of RNPs by controlling both droplet assembly and dissolution in vitro. Monotonically increasing RNA concentration initially leads to droplet assembly via complex coacervation and subsequently triggers an RNP charge inversion, which promotes disassembly. This RNA-mediated reentrant phase transition can drive the formation of dynamic droplet substructures (vacuoles) with tunable lifetimes. We propose that active cellular processes that can create an influx of RNA into RNP granules, such as transcription, can spatiotemporally control the organization and dynamics of such liquid-like organelles.

The yeast prion protein Sup35 is a translation termination factor, whose activity is modulated by sequestration into a self-perpetuating amyloid. The prion-determining domain, NM, consists of two distinct regions: an amyloidogenic N terminus domain (N) and a charged solubilizing middle region (M). To gain insight into prion conversion, we used single-molecule fluorescence resonance energy transfer (SM-FRET) and fluorescence correlation spectroscopy to investigate the structure and dynamics of monomeric NM. Low protein concentrations in these experiments prevented the formation of obligate on-pathway oligomers, allowing us to study early folding intermediates in isolation from higher-order species. SM-FRET experiments on a dual-labeled amyloid core variant (N21C/S121C, retaining wild-type prion behavior) indicated that the N region of NM adopts a collapsed form similar to ''burst-phase'' intermediates formed during the folding of many globular proteins, even though it lacks a typical hydrophobic core. The mean distance between residues 21 and 121 was Ϸ43 Å. This increased with denaturant in a noncooperative fashion to Ϸ63 Å, suggesting a multitude of interconverting species rather than a small number of discrete monomeric conformers. (1), involving self-replicating or infectious protein conformations, has attracted broad interest in recent times due to its role in the biology of debilitating neurodegenerative diseases (1, 2), protein-based inheritance of novel phenotypes in yeast (3-5), and (potentially) long-term memory (6, 7). The Saccharomyces cerevisiae translational termination factor, Sup35, is one such protein capable of switching to a self-perpetuating state. In the prion state [PSI ϩ ], the glutamine/asparagine (Q/N)-rich prion domain of Sup35 is sequestered into an amyloid conformer, reducing the efficiencies of translation termination (8). This switch causes ribosomes to read through stop codons at biologically significant rates, changing a multitude of phenotypes (9).The NM segment (253 residues) of Sup35 determines the prion state and comprises two distinct regions. The N-terminal region (residues 1-123) is abundant in uncharged polar amino acids (glutamines, asparagines, and tyrosines), and forms the major part of the amyloid core that directs the protein into the [PSI ϩ ] prion state. The highly charged middle region M (residues 124-250) confers solubility in vitro and in vivo, allowing the protein to exist in the non-prion [psi Ϫ ] state. In the prion state, the N region adopts a ␤-sheet-rich conformation, whereas the M region remains relatively unstructured (10).Structural studies on NM amyloids have provided several insights into the molecular basis of prion nucleation (11)(12)(13)(14). An early step is the establishment of an equilibrium between predominantly unstructured NM polypeptide monomers and molten oligomeric intermediates that are obligate on-pathway species (14-16). The structure and dynamics of early monomeric intermediates are of considerable interest in deciphering the molecular m...