Direct observation of tRNA-chaperoned folding of a dynamic mRNA ensemble.

T-box riboswitches are multi-domain noncoding RNAs that surveil individual amino acid availabilities in most Gram-positive bacteria. T-boxes directly bind specific tRNAs, query their aminoacylation status to detect starvation, and feedback control the transcription or translation of downstream amino-acid metabolic genes. Most T-boxes rapidly recruit their cognate tRNA ligands through an intricate three-way stem I-stem II-tRNA interaction, whose establishment is not understood. Using single-molecule FRET, SAXS, and time-resolved fluorescence, we find that the free T-box RNA assumes a broad distribution of open, semi-open, and closed conformations that only slowly interconvert. tRNA directly binds all three conformers with distinct kinetics, triggers nearly instantaneous collapses of the open conformations, and returns the T-box RNA to their pre-binding conformations upon dissociation. This scissors-like dynamic behavior is enabled by a hinge-like pseudoknot domain which poises the T-box for rapid tRNA-induced domain closure. This study reveals tRNA-chaperoned folding of flexible, multi-domain mRNAs through a Venus flytrap-like mechanism.

Single-molecule fluorescence imaging and deep learning reveal highly heterogeneous aggregation of amyloid-ß 42.

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-ß peptide (Aß42). We demonstrate that highly heterogeneous fibril formation can be quantitatively characterized in terms of the number of cross-ß 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.

Atomic view of cosolute-induced protein denaturation probed by NMR solvent paramagnetic relaxation enhancement.

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, τc 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.

Fast three-color single-molecule FRET using statistical inference.

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.

Diverse Folding Pathways of HIV-1 Protease Monomer on a Rugged Energy Landscape.

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 Förster 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.

Diffusion-limited association of disordered protein by non-native electrostatic interactions.

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.

Three-Color Single-Molecule FRET and Fluorescence Lifetime Analysis of Fast Protein Folding.

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, α3D. 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 that 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 interdye 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.

Observing Extremely Weak Protein-Protein Interactions with Conventional Single-Molecule Fluorescence Microscopy.

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.

PIF1-Interacting Transcription Factors and Their Binding Sequence Elements Determine the in Vivo Targeting Sites of PIF1.

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.

Real-time single-molecule coimmunoprecipitation of weak protein-protein interactions.

Coimmunoprecipitation (co-IP) analysis is a useful method for studying protein-protein interactions. It currently involves electrophoresis and western blotting, which are not optimized for detecting weak and transient interactions. In this protocol we describe an advanced version of co-IP analysis that uses real-time, single-molecule fluorescence imaging as its detection scheme. Bait proteins are pulled down onto the imaging plane of a total internal reflection (TIR) microscope. With unpurified cells or tissue extracts kept in reaction chambers, we observe single protein-protein interactions between the surface-immobilized bait and the fluorescent protein-labeled prey proteins in real time. Such direct recording provides an improvement of five orders of magnitude in the time resolution of co-IP analysis. With the single-molecule sensitivity and millisecond time resolution, which distinguish our method from other methods for measuring weak protein-protein interactions, it is possible to quantify the interaction kinetics and active fraction of native, unlabeled bait proteins. Real-time single-molecule co-IP analysis, which takes ∼4 h to complete from lysate preparation to kinetic analysis, provides a general avenue for revealing the rich kinetic picture of target protein-protein interactions, and it can be used, for example, to investigate the molecular lesions that drive individual cancers at the level of protein-protein interactions.

Real-time single-molecule co-immunoprecipitation analyses reveal cancer-specific Ras signalling dynamics.

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.