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Polymorphism in the structure of amyloid fibrils suggests the existence of many different assembly pathways. Characterization of this heterogeneity is the key to understanding the aggregation mechanism and toxicity, but in practice it is extremely difficult to probe individual aggregation pathways in a mixture. Here, we present development of a method combining single-molecule fluorescence lifetime imaging and deep learning for monitoring individual fibril formation in real time and their high-throughput analysis. A deep neural network (FNet) separates an image of highly overlapping fibrils into single fibril images, which allows for tracking the growth and changes in characteristics of individual fibrils. Using this method, we investigated aggregation of the 42-residue amyloid-β peptide (Aβ42). We demonstrate that highly heterogeneous fibril formation can be quantitatively characterized in terms of the number of cross-β subunits, elongation speed, growth polarity, and conformation of fibrils. Tracking individual fibril formation and growth also leads to the discovery of a general nucleation mechanism (termed heterogeneous secondary nucleation), where a fibril is formed on the surface of an oligomer with a different structure. Our development will be broadly applicable to characterization of heterogeneous aggregation processes of other proteins.

We describe theory, experiments, and analyses of three-color Förster resonance energy transfer (FRET) spectroscopy for probing sub-millisecond conformational dynamics of protein folding and binding of disordered proteins. We devise a scheme that uses single continuous-wave laser excitation of the donor instead of alternating excitation of the donor and one of the acceptors. This scheme alleviates photophysical problems of acceptors such as rapid photobleaching, which is crucial for high time resolution experiments with elevated illumination intensity. Our method exploits the molecular species with one of the acceptors absent or photobleached, from which two-color FRET data is collected in the same experiment. We show that three FRET efficiencies and kinetic parameters can be determined without alternating excitation from a global maximum likelihood analysis of two-color and three-color photon trajectories. We implement co-parallelization of CPU-GPU processing, which leads to a significant reduction of the likelihood calculation time for efficient parameter determination., Three-colour FRET is a powerful tool to study macromolecular conformational dynamics, but is temporally limited due to the experimental complexity. Here the authors develop experimental and analytical methods for probing submillisecond-time scale dynamics using single continuous-wave excitation.

The cosolvent effect arises from the interaction of cosolute molecules with a protein and alters the equilibrium between native and unfolded states. Denaturants shift the equilibrium toward the latter, while osmolytes stabilize the former. The molecular mechanism whereby cosolutes perturb protein stability is still the subject of considerable debate. Probing the molecular details of the cosolvent effect is experimentally challenging as the interactions are very weak and transient, rendering them invisible to most conventional biophysical techniques. Here, we probe cosolute–protein interactions by means of NMR solvent paramagnetic relaxation enhancement together with a formalism we recently developed to quantitatively describe, at atomic resolution, the energetics and dynamics of cosolute–protein interactions in terms of a concentration normalized equilibrium average of the interspin distance, [Formula: see text] , and an effective correlation time, τ(c). The system studied is the metastable drkN SH3 domain, which exists in dynamic equilibrium between native and unfolded states, thereby permitting us to probe the interactions of cosolutes with both states simultaneously under the same conditions. Two paramagnetic cosolute denaturants were investigated, one neutral and the other negatively charged, differing in the presence of a carboxyamide group versus a carboxylate. Our results demonstrate that attractive cosolute–protein backbone interactions occur largely in the unfolded state and some loop regions in the native state, electrostatic interactions reduce the [Formula: see text] values, and temperature predominantly impacts interactions with the unfolded state. Thus, destabilization of the native state in this instance arises predominantly as a consequence of interactions of the cosolutes with the unfolded state.

Protein aggregation is implicated as the cause of pathology in various diseases such as Alzheimer’s and Parkinson’s disease. Polymorphism in the structure of fibrils formed by aggregation suggests the existence of many different assembly pathways and therefore a heterogeneous ensemble of soluble oligomers. Characterization of this heterogeneity is the key to understanding the aggregation mechanism and toxicity of specific oligomers, but in practice it is extremely difficult because oligomers cannot be readily separated. Here, we investigate highly heterogeneous oligomerization and fibril formation of the 42-residue amyloid-β peptide (Aβ42). We developed and used new single-molecule fluorescence spectroscopic and fluorescence lifetime imaging methods, combined with deep learning for image analysis. We found that the concentration of oligomers, including dimers, is extremely low and that the dimer is conformationally diverse. Aggregation to form fibrils is also highly heterogeneous in terms of the number of strands in a fibril and the elongation speed and conformation of fibrils. This heterogeneity in all stages of aggregation explains diverse and sometimes irreproducible results of experimental studies of amyloid-β. Based on our observations and analysis, we propose a new model for aggregation of Aβ42.

We describe the theory, experiment, and analysis of three-color Förster resonance energy transfer (FRET) spectroscopy for probing conformational dynamics of a fast-folding protein, α(3)D. In three-color FRET, site-specific labeling of fluorophores is required to avoid ambiguity resulting from various species with different combinations of labeling positions. To this end, we first attached two dyes to a cysteine residue and an unnatural amino acid, and then appended a cysteine residue to the C-terminus of the protein by the sortase-mediated ligation for attaching the third dye. To determine all three FRET efficiencies, we used alternating excitation of the donor and acceptor 1 with two picosecond-pulsed lasers. Since the folded and unfolded states are not distinguishable in binned fluorescence trajectories due to fast folding on a millisecond time scale, we used a maximum likelihood method that analyzes photon trajectories without binning the data. The extracted kinetic parameters agree very well with the previously measured parameters for the same protein with two-color FRET, suggesting the addition of the third fluorophore does not affect the folding dynamics of the protein. From the extracted fractions of acceptor photon counts, the FRET efficiencies for all three dye pairs were calculated after various corrections. They were compared with the FRET efficiencies obtained from the global analysis of two-color segments collected in the same experiment. The FRET efficiencies of the folded state from the three-color segments agree with those from the two-color segments, whereas the three-color and two-color FRET efficiencies of the unfolded state are different. This happens because fluctuations of all three inter-dye distances contribute to the FRET efficiency measured in three-color FRET. We show that this difference can be accounted for by using the Gaussian chain model for the unfolded state with the parameters obtained from the analysis of two-color segments. This result shows that three-color FRET provides additional information on the flexibility of molecules that cannot be obtained from a combination of two-color FRET experiments with three dye pairs. Using the delay times of photons from the laser pulse, fluorescence lifetimes were determined using the maximum likelihood analysis. The correlation between FRET efficiencies and lifetimes of the donor, acceptor 1, and acceptor 2 was visualized in two-dimensional FRET efficiency-lifetime histograms. These histograms can be used to demonstrate the presence of conformational dynamics in a protein.

Extremely weak protein-protein interactions (PPIs), signified by micromolar or even millimolar dissociation constants, are one of the keys to understanding the rapid responses of cellular systems. Although single-molecule methods are particularly useful in determining kinetics of biological processes, their application is largely limited to rather strong interactions because of the diffraction-limited observation volume. In this study, we report a single-molecule method that allows the characterization of PPIs using a prey concentration 4 orders of magnitude lower than the dissociation constant. Instead of increasing the concentration of diffusing molecules, which is inevitably limited by the optical diffraction limit, we employed an increased density of surface bait protein. The low occupancy of the surface baits permitted determination of the kinetics with single-molecule resolution. We used this approach to study a PPI network consisting of Ras and its downstream proteins including full-length Rafs and catalytic subunits of phosphoinositide 3-kinase.

The modern energy landscape theory of protein folding predicts multiple folding pathways connecting a myriad of unfolded conformations and a well-defined folded state. However, direct experimental observation of heterogeneous folding pathways is difficult. Naturally evolved proteins typically exhibit a smooth folding energy landscape for fast and efficient folding by avoiding unfavorable kinetic traps. In this case, rapid fluctuations between unfolded conformations result in apparent two-state behavior and make different pathways indistinguishable. However, the landscape roughness can be different, depending on the selection pressures during evolution. Here, we characterize the unusually rugged folding energy landscape of human immunodeficiency virus-1 protease monomer using single-molecule Forster resonance energy transfer spectroscopy. Our data show that fluctuations between unfolded conformations are slow, which enables the experimental observation of heterogeneous folding pathways as predicted by the landscape theory. Although the landscape ruggedness is sensitive to the mutations and fluorophore locations, the folding rate is similar for various protease constructs. The natural evolution of the protease to have a rugged energy landscape likely results from intrinsic pressures to maintain robust folding when human immunodeficiency virus-1 mutates frequently, which is essential for its survival.

Intrinsically disordered proteins (IDPs) usually fold during binding to target proteins. In contrast to interactions between folded proteins, this additional folding step makes the binding process more complex. Understanding the mechanism of coupled binding and folding of IDPs requires analysis of binding pathways that involve formation of the transient complex (TC). However, experimental characterization of TC is challenging because it only appears for a very brief period during binding. Here, we use single-molecule fluorescence spectroscopy to investigate the mechanism of diffusion-limited association of an IDP. A large enhancement of the association rate is observed due to the stabilization of TC by non-native electrostatic interactions. Moreover, photon-by-photon analysis reveals that the lifetime of TC for IDP binding is at least two orders of magnitude longer than that for binding of two folded proteins. This result suggests the long lifetime of TC is generally required for folding of IDPs during binding processes., Intrinsically disordered proteins (IDPs) usually fold during binding to target proteins which involves the formation of a transient complex (TC). Here authors use single-molecule FRET to show that the lifetime of TC for IDP binding is very long due to the stabilization by non-native electrostatic interactions, which makes fast association possible.

The bHLH transcription factor PHYTOCHROME INTERACTING FACTOR1 (PIF1) binds G-box elements in vitro and inhibits light-dependent germination in Arabidopsis thaliana. A previous genome-wide analysis of PIF1 targeting indicated that PIF1 binds 748 sites in imbibed seeds, only 59% of which possess G-box elements. This suggests the G-box is not the sole determinant of PIF1 targeting. The targeting of PIF1 to specific sites could be stabilized by PIF1-interacting transcription factors (PTFs) that bind other nearby sequence elements. Here, we report PIF1 targeting sites are enriched with not only G-boxes but also with other hexameric sequence elements we named G-box coupling elements (GCEs). One of these GCEs possesses an ACGT core and serves as a binding site for group A bZIP transcription factors, including ABSCISIC ACID INSENSITIVE5 (ABI5), which inhibits seed germination in abscisic acid signaling. PIF1 interacts with ABI5 and other group A bZIP transcription factors and together they target a subset of PIF1 binding sites in vivo. In vitro single-molecule fluorescence imaging confirms that ABI5 facilitates PIF1 binding to DNA fragments possessing multiple G-boxes or the GCE alone. Thus, we show in vivo PIF1 targeting to specific binding sites is determined by its interaction with PTFs and their binding to GCEs.

The conventional co-immunoprecipitation provides static and qualitative information about protein-protein interactions. Lee et al. report real-time imaging of co-immunoprecipitation process with single-molecule resolution, allowing for characterization of the native Ras proteins derived from individual cancers.

Co-immunoprecipitation (co-IP) has become a standard technique, but its protein-band output provides only static, qualitative information about protein–protein interactions. Here we demonstrate a real-time single-molecule co-IP technique that generates real-time videos of individual protein–protein interactions as they occur in unpurified cell extracts. By analysing single Ras–Raf interactions with a 50-ms time resolution, we have observed transient intermediates of the protein–protein interaction and determined all the essential kinetic rates. Using this technique, we have quantified the active fraction of native Ras proteins in xenograft tumours, normal tissue and cancer cell lines. We demonstrate that the oncogenic Ras mutations selectively increase the active-Ras fraction by one order of magnitude, without affecting total Ras levels or single-molecule signalling kinetics. Our approach allows us to probe the previously hidden, dynamic aspects of weak protein–protein interactions. It also suggests a path forward towards precision molecular diagnostics at the protein–protein interaction level., Co-immunoprecipitation provides static and qualitative information about protein–protein interactions. Lee et al. create real-time movies of single protein–protein interactions during co-immunoprecipitation, and use them to assess the dynamics of mutant Ras proteins derived from tumours.

Co-immunoprecipitation (co-IP) and western blotting are the gold standards for assessing protein-protein interactions. However, their protein band output only provides qualitative and static information on protein compositions and their interactions. In this study, we demonstrate a quantitative transformation of the traditional western and co-ip techniques using in situ single-molecule level measurements with millisecond time resolution. We have quantified the molar concentration of specific proteins in whole cell or tissue extracts and determined the stoichiometry and kinetics of their interactions. We have then used our methods to quantify the expression level and signaling frequency of oncogenic Ras that has been directly pulled down from tumor tissues. Our results suggest that these techniques will make valuable additions to the molecular diagnostic repertoire of modern biomedicine.Remark to the program chairsRecently, Dr. Taekjip Ha and co-workers reported on single-molecule pull down. They showed that target proteins of tiny amount can be selectively immobilized on surface while thousands of other proteins are effectively rejected from the same surface (Nature 473, 484 (2011)). Inspired by this approach, we have transformed the co-IP and western blotting into quantitative methods. By employing fluorescently labeled binding competitors, we demonstrate single-molecule western analysis, in which we robustly measure the true molar concentration of specific proteins in cell or tissue extracts. We also demonstrate that in situ imaging can be done during single-molecule co-IP, which crucially enables to access weak and transient protein-protein interactions. Finally, we show that oncogenic Ras proteins pulled down from a tumor tissue indeed shows a higher activation percentage as well as a higher expression level than Ras from normal tissues. Our techniques rapidly assess the strength of a certain signaling step, which should be useful for the molecular diagnostics.

As a major protein detection system, western blot has commonly used to probe changes in amount of total or activated proteins in the cell. In this technique, antibodies that are specific to target proteins are recruited as to measure protein amount in the cell extract. Nevertheless, as the detection level of the western blot changes depending on antibody efficacy and differences in individual systems, protein amount has been obtained only in qualitative manner. In this study, we introduce Single Molecule Western Blot(SMW) assay that incorporates conventional western blot system into single-molecule fluorescence microscopy, providing expression levels of proteins using whole cell extract. Our results demonstrate the use of this technique into a cell signaling system suggesting its potency for future biomedical research and diagnostics.

Pore expansion, an essential step for SNARE-mediated membrane fusion, has not been well studied due to the lack of a reliable content mixing assay. Recently, we have developed a new assay to detect the inter-vesicular mixing of large cargoes at the size of several nanometers at the single molecule and vesicle level [1]. Through our new assay, we found that the neuronal SNARE complexes alone, without regulatory proteins, are able to expand fusion pore inefficiently. By interacting with t-SNARE proteins through a trans-conformer [2], membrane anchor synaptotagmin 1 and Ca2+ facilitate SNARE complex formation for fusion pore expansion.References[1] Diao, J., Su, Z., Ishitsuka, Y., Lu, B., Lee, K.S., Lai, Y., Shin, Y.-K. & Ha, T Nat. Commun.1, 54 (2010).[2] Lee, H.-K., Yang, Y., Su, Z., Hyeon, C., Lee, T.-S., Lee, H.-W., Kweon, D.-H., Shin, Y.-K. & Yoon, T.-Y. Science 328, 760-763 (2010).