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 FRET and molecular diffusion analysis characterize stable oligomers of amyloid-ß 42 of extremely low population.

Soluble oligomers produced during protein aggregation have been thought to be toxic species causing various diseases. Characterization of these oligomers is difficult because oligomers are a heterogeneous mixture, which is not readily separable, and may appear transiently during aggregation. Single-molecule spectroscopy can provide valuable information by detecting individual oligomers, but there have been various problems in determining the size and concentration of oligomers. In this work, we develop and use a method that analyzes single-molecule fluorescence burst data of freely diffusing molecules in solution based on molecular diffusion theory and maximum likelihood method. We demonstrate that the photon count rate, diffusion time, population, and Förster resonance energy transfer (FRET) efficiency can be accurately determined from simulated data and the experimental data of a known oligomerization system, the tetramerization domain of p53. We used this method to characterize the oligomers of the 42-residue amyloid-ß (Aß42) peptide. Combining peptide incubation in a plate reader and single-molecule free-diffusion experiments allows for the detection of stable oligomers appearing at various stages of aggregation. We find that the average size of these oligomers is 70-mer and their overall population is very low, less than 1 nM, in the early and middle stages of aggregation of 1 µM Aß42 peptide. Based on their average size and long diffusion time, we predict the oligomers have a highly elongated rod-like shape.

Analysis of photon trajectories from diffusing single molecules.

In single-molecule free diffusion experiments, molecules spend most of the time outside a laser spot and generate bursts of photons when they diffuse through the focal spot. Only these bursts contain meaningful information and, therefore, are selected using physically reasonable criteria. The analysis of the bursts must take into account the precise way they were chosen. We present new methods that allow one to accurately determine the brightness and diffusivity of individual molecule species from the photon arrival times of selected bursts. We derive analytical expressions for the distribution of inter-photon times (with and without burst selection), the distribution of the number of photons in a burst, and the distribution of photons in a burst with recorded arrival times. The theory accurately treats the bias introduced due to the burst selection criteria. We use a Maximum Likelihood (ML) method to find the molecule's photon count rate and diffusion coefficient from three kinds of data, i.e., the bursts of photons with recorded arrival times (burstML), inter-photon times in bursts (iptML), and the numbers of photon counts in a burst (pcML). The performance of these new methods is tested on simulated photon trajectories and on an experimental system, the fluorophore Atto 488.

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.

Theory and Analysis of Single-Molecule FRET Experiments.

Inter-dye distances and conformational dynamics can be studied using single-molecule FRET measurements. We consider two approaches to analyze sequences of photons with recorded photon colors and arrival times. The first approach is based on FRET efficiency histograms obtained from binned photon sequences. The experimental histograms are compared with the theoretical histograms obtained using the joint distribution of acceptor and donor photons or the Gaussian approximation. In the second approach, a photon sequence is analyzed without binning. The parameters of a model describing conformational dynamics are found by maximizing the appropriate likelihood function. The first approach is simpler, while the second one is more accurate, especially when the population of species is small and transition rates are fast. The likelihood-based analysis as well as the recoloring method has the advantage that diffusion of molecules through the laser focus can be rigorously handled.

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.

Disordered proteins follow diverse transition paths as they fold and bind to a partner.

Transition paths of macromolecular conformational changes such as protein folding are predicted to be heterogeneous. However, experimental characterization of the diversity of transition paths is extremely challenging because it requires measuring more than one distance during individual transitions. In this work, we used fast three-color single-molecule Förster resonance energy transfer spectroscopy to obtain the distribution of binding transition paths of a disordered protein. About half of the transitions follow a path involving strong non-native electrostatic interactions, resulting in a transition time of 300 to 800 microseconds. The remaining half follow more diverse paths characterized by weaker electrostatic interactions and more than 10 times shorter transition path times. The chain flexibility and non-native interactions make diverse binding pathways possible, allowing disordered proteins to bind faster than folded proteins.

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.

Probing the mechanism of inhibition of amyloid-ß(1-42)-induced neurotoxicity by the chaperonin GroEL.

The human chaperonin Hsp60 is thought to play a role in the progression of Alzheimer's disease by mitigating against intracellular ß-amyloid stress. Here, we show that the bacterial homolog GroEL (51% sequence identity) reduces the neurotoxic effects of amyloid-ß(1-42) (Aß42) on human neural stem cell-derived neuronal cultures. To understand the mechanism of GroEL-mediated abrogation of neurotoxicity, we studied the interaction of Aß42 with GroEL using a variety of biophysical techniques. Aß42 binds to GroEL as a monomer with a lifetime of ∼1 ms, as determined from global analysis of multiple relaxation-based NMR experiments. Dynamic light scattering demonstrates that GroEL dissolves small amounts of high-molecular-weight polydisperse aggregates present in fresh soluble Aß42 preparations. The residue-specific transverse relaxation rate profile for GroEL-bound Aß42 reveals the presence of three anchor-binding regions (residues 16-21, 31-34, and 40-41) located within the hydrophobic GroEL-consensus binding sequences. Single-molecule FRET analysis of Aß42 binding to GroEL results in no significant change in the FRET efficiency of a doubly labeled Aß42 construct, indicating that Aß42 samples a random coil ensemble when bound to GroEL. Finally, GroEL substantially slows down the disappearance of NMR visible Aß42 species and the appearance of Aß42 protofibrils and fibrils as monitored by electron and atomic force microscopies. The latter observations correlate with the effect of GroEL on the time course of Aß42-induced neurotoxicity. These data provide a physical basis for understanding how Hsp60 may serve to slow down the progression of Alzheimer's disease.

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.

Highly Disordered Amyloid-ß Monomer Probed by Single-Molecule FRET and MD Simulation.

Monomers of amyloid-ß (Aß) protein are known to be disordered, but there is considerable controversy over the existence of residual or transient conformations that can potentially promote oligomerization and fibril formation. We employed single-molecule Förster resonance energy transfer (FRET) spectroscopy with site-specific dye labeling using an unnatural amino acid and molecular dynamics simulations to investigate conformations and dynamics of Aß isoforms with 40 (Aß40) and 42 residues (Aß42). The FRET efficiency distributions of both proteins measured in phosphate-buffered saline at room temperature show a single peak with very similar FRET efficiencies, indicating there is apparently only one state. 2D FRET efficiency-donor lifetime analysis reveals, however, that there is a broad distribution of rapidly interconverting conformations. Using nanosecond fluorescence correlation spectroscopy, we measured the timescale of the fluctuations between these conformations to be ∼35 ns, similar to that of disordered proteins. These results suggest that both Aß40 and Aß42 populate an ensemble of rapidly reconfiguring unfolded states, with no long-lived conformational state distinguishable from that of the disordered ensemble. To gain molecular-level insights into these observations, we performed molecular dynamics simulations with a force field optimized to describe disordered proteins. We find, as in experiments, that both peptides populate configurations consistent with random polymer chains, with the vast majority of conformations lacking significant secondary structure, giving rise to very similar ensemble-averaged FRET efficiencies.

Transition Path Times Measured by Single-Molecule Spectroscopy.

The transition path is a tiny fraction of a molecular trajectory during which the free-energy barrier is crossed. It is a single-molecule property and contains all mechanistic information of folding processes of biomolecules such as proteins and nucleic acids. However, the transition path has been difficult to probe because it is short and rarely visited when transitions actually occur. Recent technical advances in single-molecule spectroscopy have made it possible to directly probe transition paths, which has opened up new theoretical and experimental approaches to investigating folding mechanisms. This article reviews recent single-molecule fluorescence and force spectroscopic measurements of transition path times and their connection to both theory and simulations.

Protein folding transition path times from single molecule FRET.

The transition path is the tiny segment of a single molecule trajectory when the free energy barrier between states is crossed and for protein folding contains all of the information about the self-assembly mechanism. As a first step toward obtaining structural information during the transition path from experiments, single molecule FRET spectroscopy has been used to determine average transition path times from a photon-by-photon analysis of fluorescence trajectories. These results, obtained for several different proteins, have already provided new and demanding tests that support both the accuracy of all-atom molecular dynamics simulations and the basic postulates of energy landscape theory of protein folding.

Oligomerization of the tetramerization domain of p53 probed by two- and three-color single-molecule FRET.

We describe a method that combines two- and three-color single-molecule FRET spectroscopy with 2D FRET efficiency-lifetime analysis to probe the oligomerization process of intrinsically disordered proteins. This method is applied to the oligomerization of the tetramerization domain (TD) of the tumor suppressor protein p53. TD exists as a monomer at subnanomolar concentrations and forms a dimer and a tetramer at higher concentrations. Because the dissociation constants of the dimer and tetramer are very close, as we determine in this paper, it is not possible to characterize different oligomeric species by ensemble methods, especially the dimer that cannot be readily separated. However, by using single-molecule FRET spectroscopy that includes measurements of fluorescence lifetime and two- and three-color FRET efficiencies with corrections for submillisecond acceptor blinking, we show that it is possible to obtain structural information for individual oligomers at equilibrium and to determine the dimerization kinetics. From these analyses, we show that the monomer is intrinsically disordered and that the dimer conformation is very similar to that of the tetramer but the C terminus of the dimer is more flexible.

Analysis of Fluorescence Lifetime and Energy Transfer Efficiency in Single-Molecule Photon Trajectories of Fast-Folding Proteins.

In single-molecule Förster resonance energy transfer (FRET) spectroscopy, the dynamics of molecular processes are usually determined by analyzing the fluorescence intensity of donor and acceptor dyes. Since FRET efficiency is related to fluorescence lifetimes, additional information can be extracted by analyzing fluorescence intensity and lifetime together. For fast processes where individual states are not well separated in a trajectory, it is not easy to obtain the lifetime information. Here, we present analysis methods to utilize fluorescence lifetime information from single-molecule FRET experiments, and apply these methods to three fast-folding, two-state proteins. By constructing 2D FRET efficiency-lifetime histograms, the correlation can be visualized between the FRET efficiency and fluorescence lifetimes in the presence of the submicrosecond to millisecond dynamics. We extend the previously developed method for analyzing delay times of donor photons to include acceptor delay times. To determine the kinetics and lifetime parameters accurately, we used a maximum likelihood method. We found that acceptor blinking can lead to inaccurate parameters in the donor delay time analysis. This problem can be solved by incorporating acceptor blinking into a model. While the analysis of acceptor delay times is not affected by acceptor blinking, it is more sensitive to the shape of the delay time distribution resulting from a broad conformational distribution in the unfolded state.

Structural origin of slow diffusion in protein folding.

Experimental, theoretical, and computational studies of small proteins suggest that interresidue contacts not present in the folded structure play little or no role in the self-assembly mechanism. Non-native contacts can, however, influence folding kinetics by introducing additional local minima that slow diffusion over the global free-energy barrier between folded and unfolded states. Here, we combine single-molecule fluorescence with all-atom molecular dynamics simulations to discover the structural origin for the slow diffusion that markedly decreases the folding rate for a designed α-helical protein. Our experimental determination of transition path times and our analysis of the simulations point to non-native salt bridges between helices as the source, which provides a quantitative glimpse of how specific intramolecular interactions influence protein folding rates by altering dynamics and not activation free energies.

Testing Landscape Theory for Biomolecular Processes with Single Molecule Fluorescence Spectroscopy.

Although Kramers' theory for diffusive barrier crossing on a 1D free energy profile plays a central role in landscape theory for complex biomolecular processes, it has not yet been rigorously tested by experiment. Here we test this 1D diffusion scenario with single molecule fluorescence measurements of DNA hairpin folding. We find an upper bound of 2.5 µs for the average transition path time, consistent with the predictions by theory with parameters determined from optical tweezer measurements.