Single-molecule FRET Spectroscopy Research Articles

Enzyme activation by urea reveals the interplay between conformational dynamics and substrate binding: a single-molecule FRET study.

Proteins often harness extensive motions of domains and subunits to promote their function. Deciphering how these movements impact activity is key for understanding life's molecular machinery. The enzyme adenylate kinase is an intriguing example for this relationship; it ensures efficient catalysis by large-scale domain motions that lead to the enclosure of the bound substrates ATP and AMP. At high concentrations, AMP also operates as an allosteric inhibitor of the protein. Surprisingly, the enzyme is activated by urea, a compound commonly acting as a denaturant. Combining single-molecule FRET spectroscopy and enzymatic activity studies, we find that urea interferes with two key mechanisms that contribute to enzyme efficacy. First, urea promotes the open conformation of the enzyme, aiding the proper positioning of the substrates. Second, urea decreases AMP affinity, paradoxically facilitating a more efficient progression towards the catalytically active complex. These results signify the important interplay between conformational dynamics and chemical steps, including binding, in the activity of enzymes. State-of-the-art tools, such as single-molecule fluorescence spectroscopy, offer new insights into how enzymes balance different conformations to regulate activity.

Single-Molecule Characterization of Heterogeneous Oligomer Formation during Co-Aggregation of 40- and 42-Residue Amyloid-β.

The two most abundant isoforms of amyloid-β (Aβ) are the 40- (Aβ40) and 42-residue (Aβ42) peptides. Since they coexist and there is a correlation between toxicity and the ratio of the two isoforms, quantitative characterization of their interactions is crucial for understanding the Aβ aggregation mechanism. In this work, we follow the aggregation of individual isoforms in a mixture using single-molecule FRET spectroscopy by labeling Aβ42 and Aβ40 with the donor and acceptor fluorophores, respectively. We found that there are two phases of aggregation. The first phase consists of coaggregation of Aβ42 with a small amount of Aβ40, while the second phase results mostly from aggregation of Aβ40. We also found that the aggregation of Aβ42 is slowed by Aβ40 while the aggregation of Aβ40 is accelerated by Aβ42 in a concentration-dependent manner. The formation of oligomers was monitored by incubating mixtures in a plate reader and performing a single-molecule free-diffusion experiment at several different stages of aggregation. The detailed properties of the oligomers were obtained by maximum likelihood analysis of fluorescence bursts. The FRET efficiency distribution is much broader than that of the Aβ42 oligomers, indicating the diversity in isoform composition of the oligomers. Pulsed interleaved excitation experiments estimate that the fraction of Aβ40 in the co-oligomers in a 1:1 mixture of Aβ42 and Aβ40 varies between 0 and 20%. The detected oligomers were mostly co-oligomers especially at the physiological ratio of Aβ42 and Aβ40 (1:10), suggesting the critical role of Aβ40 in oligomer formation and aggregation.

Partial wrapping of single-stranded DNA by replication protein A and modulation through phosphorylation.

Single-stranded DNA (ssDNA) intermediates which emerge during DNA metabolic processes are shielded by replication protein A (RPA). RPA binds to ssDNA and acts as a gatekeeper to direct the ssDNA towards downstream DNA metabolic pathways with exceptional specificity. Understanding the mechanistic basis for such RPA-dependent functional specificity requires knowledge of the structural conformation of ssDNA when RPA-bound. Previous studies suggested a stretching of ssDNA by RPA. However, structural investigations uncovered a partial wrapping of ssDNA around RPA. Therefore, to reconcile the models, in this study, we measured the end-to-end distances of free ssDNA and RPA-ssDNA complexes using single-molecule FRET and double electron-electron resonance (DEER) spectroscopy and found only a small systematic increase in the end-to-end distance of ssDNA upon RPA binding. This change does not align with a linear stretching model but rather supports partial wrapping of ssDNA around the contour of DNA binding domains of RPA. Furthermore, we reveal how phosphorylation at the key Ser-384 site in the RPA70 subunit provides access to the wrapped ssDNA by remodeling the DNA-binding domains. These findings establish a precise structural model for RPA-bound ssDNA, providing valuable insights into how RPA facilitates the remodeling of ssDNA for subsequent downstream processes.

DNA binding redistributes activation domain ensemble and accessibility in pioneer factor Sox2

More than 1600 human transcription factors orchestrate the transcriptional machinery to control gene expression and cell fate. Their function is conveyed through intrinsically disordered regions (IDRs) containing activation or repression domains but lacking quantitative structural ensemble models prevents their mechanistic decoding. Here we integrate single-molecule FRET and NMR spectroscopy with molecular simulations showing that DNA binding can lead to complex changes in the IDR ensemble and accessibility. The C-terminal IDR of pioneer factor Sox2 is highly disordered but its conformational dynamics are guided by weak and dynamic charge interactions with the folded DNA binding domain. Both DNA and nucleosome binding induce major rearrangements in the IDR ensemble without affecting DNA binding affinity. Remarkably, interdomain interactions are redistributed in complex with DNA leading to variable exposure of two activation domains critical for transcription. Charged intramolecular interactions allowing for dynamic redistributions may be common in transcription factors and necessary for sensitive tuning of structural ensembles.

Fast dynamics shape the function of the AAA+ machine ClpB: Lessons from single-molecule FRET spectroscopy

Fast dynamics shape the function of the AAA+ machine ClpB: Lessons from single-molecule FRET spectroscopy

Single-molecule FRET probes allosteric effects on protein-translocating pore loops of a AAA+ machine

AAA+ proteins (ATPases associated with various cellular activities) comprise a family of powerful ring-shaped ATP-dependent translocases that carry out numerous vital substrate-remodeling functions. ClpB is a AAA+ protein disaggregation machine that forms a two-tiered hexameric ring, with flexible pore loops protruding into its center and binding to substrate proteins. It remains unknown whether these pore loops contribute only passively to substrate-protein threading or have a more active role. Recently, we have applied single-molecule FRET spectroscopy to directly measure the dynamics of substrate-binding pore loops in ClpB. We have reported that the three pore loops of ClpB (PL1-3) undergo large-scale fluctuations on the microsecond timescale that are likely to be mechanistically important for disaggregation. Here, using single-molecule FRET, we study the allosteric coupling between the pore loops and the two nucleotide-binding domains of ClpB (NBD1–2). By mutating the conserved Walker B motifs within the NBDs to abolish ATP hydrolysis, we demonstrate how the nucleotide state of each NBD tunes pore-loop dynamics. This effect is surprisingly long-ranged; in particular, PL2 and PL3 respond differentially to a Walker B mutation in either NBD1 or NBD2, as well as to mutations in both. We characterize the conformational dynamics of pore loops and the allosteric paths connecting NBDs to pore loops by molecular dynamics simulations and find that both principal motions and allosteric paths can be altered by changing the ATPase state of ClpB. Remarkably, PL3, which is highly conserved in AAA+ machines, is found to favor an upward conformation when only NBD1 undergoes ATP hydrolysis but a downward conformation when NBD2 is active. These results explicitly demonstrate a significant long-range allosteric effect of ATP hydrolysis sites on pore-loop dynamics. Pore loops are therefore established as active participants that undergo ATP-dependent conformational changes to translocate substrate proteins through the central pores of AAA+ machines.

Single-molecule-binding studies of antivirals targeting the hepatitis C virus core protein.

The hepatitis C virus is associated with nearly 300,000 deaths annually. At the core of the virus is an RNA-protein complex called the nucleocapsid, which consists of the viral genome and many copies of the core protein. Because the assembly of the nucleocapsid is a critical step in viral replication, a considerable amount of effort has been devoted to identifying antiviral therapeutics that can bind to the core protein and disrupt assembly. Although several candidates have been identified, little is known about how they interact with the core protein or how those interactions alter the structure and thus the function of this viral protein. Our work biochemically characterizes several of these binding interactions, highlighting both similarities and differences as well as strengths and weaknesses. These insights bolster the notion that this viral protein is a viable target for novel therapeutics and will help to guide future developments of these candidate antivirals.

Allosteric communication between ligand binding domains modulates substrate inhibition in adenylate kinase

Enzymes play a vital role in life processes; they control chemical reactions and allow functional cycles to be synchronized. Many enzymes harness large-scale motions of their domains to achieve tremendous catalytic prowess and high selectivity for specific substrates. One outstanding example is provided by the three-domain enzyme adenylate kinase (AK), which catalyzes phosphotransfer between ATP to AMP. Here we study the phenomenon of substrate inhibition by AMP and its correlation with domain motions. Using single-molecule FRET spectroscopy, we show that AMP does not block access to the ATP binding site, neither by competitive binding to the ATP cognate site nor by directly closing the LID domain. Instead, inhibitory concentrations of AMP lead to a faster and more cooperative domain closure by ATP, leading in turn to an increased population of the closed state. The effect of AMP binding can be modulated through mutations throughout the structure of the enzyme, as shown by the screening of an extensive AK mutant library. The mutation of multiple conserved residues reduces substrate inhibition, suggesting that substrate inhibition is an evolutionary well conserved feature in AK. Combining these insights, we developed a model that explains the complex activity of AK, particularly substrate inhibition, based on the experimentally observed opening and closing rates. Notably, the model indicates that the catalytic power is affected by the microsecond balance between the open and closed states of the enzyme. Our findings highlight the crucial role of protein motions in enzymatic activity.

Refining HIV envelope protein conformational data from different experimental sources using bias-resampling methods and information theory.

Human immunodeficiency virus (HIV) remains a global epidemic, and vaccine development has been challenging. This challenge is due in part to conflicting data regarding which conformational states of the envelope glycoprotein (Env) are effectively targeted by neutralizing antibodies. Env is known to undergo conformational change as part of its function. Identifying the conformations best bound by neutralizing antibodies will improve vaccine design. To this end, we use bias resampling ensemble refinement (BRER) to integrate and compare prior spectroscopic measurements of HIV Env conformation. By using both electron paramagnetic resonance and single-molecule FRET data as restraints in conformational ensemble refinement, we generate models for the sets of HIV Env conformations consistent with each set of measurements. Results show that the “hidden” conformational states identified via single-molecule FRET spectroscopy indeed show differences from reference cryo-EM structures. Further analysis shows that the “hidden” conformational states have different distance distributions for some residue pairs than those measured via DEER. We have developed information-theoretic techniques to identify DEER label sites that would best differentiate conformational states defined by single-molecule FRET. We hope that testing of these proposed DEER experiments will help resolve the discrepancy between observations using different spectroscopic techniques and better elucidate the conformational heterogeneity of HIV Env. Extension to datasets in the presence of bound neutralizing antibodies will help elucidate the key conformations recognized by broadly neutralizing anti-HIV antibodies and, we hope, further assist in vaccine design.

Resolving the kinetics of transcription factor recognition of the target DNA site via advanced single-molecule FRET spectroscopy.

Transcription factors function by specifically binding to target DNA sites to initiate gene expression. Such DNA recognition process usually involves an interplay between high-affinity binding to the cognate sequence and low-affinity binding to any other DNA. The latter enables scanning of the DNA via a 1D diffusive process, which combined with 3D diffusion-collision kinetics, facilitates target location. 1D diffusion has been observed using single-molecule fluorescence tracking on many DNA binding proteins (DPBs). Powerful experimental methods are also available to determine the sequence logo recognized by any given DBP. However, there are no approaches available that resolve the kinetics of locking into the target site, which is essential to elucidate the mechanisms of cognate DNA recognition. Here we introduce such an approach, by using single-molecule FRET spectroscopy and maximum likelihood analysis of photon arrival times. We apply it to determine the rates of lock-into and release-from the target site of the Engrailed homeodomain (EnHD). To resolve between non-specific and cognate DNA binding we exploit the recent discovery that EnHD changes conformation upon cognate binding, resulting in a large shift in transfer efficiency between a donor-acceptor pair placed at the protein ends. Our experiments on a 21-bp dsDNA containing the EnHD cognate sequence reveal that EnHD finds its target in few milliseconds. Remarkably, the on-rate at the C50 is mostly invariant over orders of magnitude differences in ionic strength and DNA concentration. This is because the changes in affinity caused by ionic strength are mostly recapitulated on the off-rate, which is the exact opposite to diffusion-control kinetics. We hence conclude that EnHD's lock-into target kinetics are controlled by its conformational dynamics, which makes the response time for controlling transcription only dependent on site occupancy levels.

Inhibited complete folding of consecutive human telomeric G-quadruplexes.

Noncanonical DNA structures, termed G-quadruplexes, are present in human genomic DNA and are important elements in many DNA metabolic processes. Multiple sites in the human genome have G-rich DNA stretches able to support formation of several consecutive G-quadruplexes. One of those sites is the telomeric overhang region that has multiple repeats of TTAGGG and is tightly associated with both cancer and aging. We investigated the folding of consecutive G-quadruplexes in both potassium- and sodium-containing solutions using single-molecule FRET spectroscopy, circular dichroism, thermal melting and molecular dynamics simulations. Our observations show coexistence of partially and fully folded DNA, the latter consisting of consecutive G-quadruplexes. Following the folding process over hours in sodium-containing buffers revealed fast G-quadruplex folding but slow establishment of thermodynamic equilibrium. We find that full consecutive G-quadruplex formation is inhibited by the many DNA structures randomly nucleating on the DNA, some of which are off-path conformations that need to unfold to allow full folding. Our study allows describing consecutive G-quadruplex formation in both nonequilibrium and equilibrium conditions by a unified picture, where, due to the many possible DNA conformations, full folding with consecutive G-quadruplexes as beads on a string is not necessarily achieved.

Ligand-Dependent Volumetric Characterization of Manganese Riboswitch Folding: A High-Pressure Single-Molecule Kinetic Study.

Nanoscopic differences in free volume result in pressure-dependent changes in free energies which can therefore impact folding/unfolding stability of biomolecules. Although such effects are typically insignificant under ambient pressure conditions, they are crucially important for deep ocean marine life, where the hydraulic pressure can be on the kilobar scale. In this work, single molecule FRET spectroscopy is used to study the effects of pressure on both the kinetics and overall thermodynamics for folding/unfolding of the manganese riboswitch. Detailed pressure-dependent analysis of the conformational kinetics allows one to extract precision changes (σ ≲ 4-8 Å3) in free volumes not only between the fully folded/unfolded conformations but also with respect to the folding transition state of the manganese riboswitch. This permits first extraction of a novel "reversible work" free energy (PΔV) landscape, which reveals a monotonic increase in manganese riboswitch volume along the folding coordinate. Furthermore, such a tool permits exploration of pressure-dependent effects on both Mn2+ binding and riboswitch folding, which demonstrate that ligand attachment stabilizes the riboswitch under pressure by decreasing the volume increase upon folding (ΔΔV < 0). Such competition between ligand binding and pressure-induced denaturation dynamics could be of significant evolutionary advantage, compensating for a weakening in riboswitch tertiary structure with pressure-mediated ligand binding and promotion of folding response.

Fast dynamics shape the function of the AAA+ machine ClpB: lessons from single-molecule FRET spectroscopy.

It has been recently shown that in some proteins, tertiary-structure dynamics occur surprisingly fast, that is on the microsecond or sub-millisecond time scales. In this State of the Art Review, we discuss how such ultrafast domain motions relate to the function of caseinolytic peptidase B (ClpB), a AAA+ disaggregation machine. ClpB is a large hexameric protein that collaborates with cellular chaperone machinery to rescue protein chains from aggregates. We used single-molecule FRET spectroscopy to capture the dynamics of essential structural elements within this machine. It was found that the middle domain of ClpB, known to act as its activator, toggles between two states much faster than the overall activity cycle of the protein, suggesting a novel mode of continuous, tunable switching. Motions of the N-terminal domain were observed to restrict the conformational space of the M domain in the absence of a substrate protein, thereby preventing it from tilting and spuriously activating ClpB. Finally, microsecond dynamics of pore loops responsible for substrate pulling through ClpB's central channel, together with their response to specific perturbations, point to a Brownian-ratchet mechanism for protein translocation. Based on our findings, we propose a two-time-scale model for the activity of ClpB, in which fast conformational dynamics affect slower functional steps, determined by ATP hydrolysis time. Future work on this and other proteins is likely to shed further light on the role of ultrafast dynamics on protein function.

Combining Coarse-Grained Simulations and Single Molecule Analysis Reveals a Three-State Folding Model of the Guanidine-II Riboswitch.

Riboswitch RNAs regulate gene expression by conformational changes induced by environmental conditions and specific ligand binding. The guanidine-II riboswitch is proposed to bind the small molecule guanidinium and to subsequently form a kissing loop interaction between the P1 and P2 hairpins. While an interaction was shown for isolated hairpins in crystallization and electron paramagnetic resonance experiments, an intrastrand kissing loop formation has not been demonstrated. Here, we report the first evidence of this interaction in cis in a ligand and Mg2+ dependent manner. Using single-molecule FRET spectroscopy and detailed structural information from coarse-grained simulations, we observe and characterize three interconvertible states representing an open and kissing loop conformation as well as a novel Mg2+ dependent state for the guanidine-II riboswitch from E. coli. The results further substantiate the proposed switching mechanism and provide detailed insight into the regulation mechanism for the guanidine-II riboswitch class. Combining single molecule experiments and coarse-grained simulations therefore provides a promising perspective in resolving the conformational changes induced by environmental conditions and to yield molecular insights into RNA regulation.

Conformational Expansion of Tau in Condensates Promotes Irreversible Aggregation.

Liquid-liquid phase separation (LLPS) of proteins into biomolecular condensates has emerged as a fundamental principle underpinning cellular function and malfunction. Indeed, many human pathologies, including protein misfolding diseases, are linked to aberrant liquid-to-solid phase transitions, and disease-associated protein aggregates often nucleate through phase separation. The molecular level determinants that promote pathological phase transitions remain, however, poorly understood. Here we study LLPS of the microtubule-associated protein Tau, whose aberrant aggregation is associated with a number of neurodegenerative diseases, including Alzheimer's disease. Using single molecule spectroscopy, we probe directly the conformational changes that the protein undergoes as a result of LLPS. We perform single-molecule FRET and fluorescence correlation spectroscopy experiments to monitor the intra- and intermolecular changes and demonstrate that the N- and C-terminal regions of Tau become extended, thus exposing the microtubule-binding region. These changes facilitate intermolecular interactions and allow for the formation of nanoscale clusters of Tau. Our results suggest that these clusters can promote the fibrillization of Tau, which can be dramatically accelerated by disease-related mutations P301L and P301S. Our findings thus provide important molecular insights into the mechanism of protein phase separation and the conversion of protein condensates from functional liquid assemblies to pathological aggregates.

Smaller molecules crowd better: Crowder size dependence revealed by single-molecule FRET studies and depletion force modeling analysis.

The cell is an extremely crowded environment, which is known to have a profound impact on the thermodynamics, functionality, and conformational stability of biomolecules. Speculations from recent theoretical molecular dynamics studies suggest an intriguing size dependence to such purely entropic crowding effects, whereby small molecular weight crowders under constant enthalpy conditions are more effective than larger crowders on a per volume basis. If experimentally confirmed, this would be profoundly significant, as the cellular cytoplasm is also quite concentrated in smaller molecular weight solutes such as inorganic ions, amino acids, and various metabolites. The challenge is to perform such studies isolating entropic effects under isoenthalpic conditions. In this work, we first present results from single-molecule FRET spectroscopy (smFRET) on the molecular size-dependent crowding stabilization of a simple RNA tertiary motif (the GAAA tetraloop-tetraloop receptor), indeed providing evidence in support of the surprising notion in the crowding literature that "smaller is better." Specifically, systematic smFRET studies as a function of crowder solute size reveal that smaller molecules both significantly increase the RNA tertiary folding rate and, yet, simultaneously decrease the unfolding rate, predicting strongly size-dependent stabilization of RNA tertiary structures under crowded cellular conditions. The size dependence of these effects has been explored via systematic variation of crowder size over a broad range of molecular weights (90-3000 amu). Furthermore, corresponding temperature dependent studies indicate the systematic changes in the folding equilibrium to be predominantly entropic in origin, i.e., consistent with a fundamental picture of entropic molecular crowding without additional enthalpic interactions. Most importantly, all trends in the single-molecule crowding data can be quantitatively recapitulated by a simple analytic depletion force model, whereby excluded volume interactions represent the major thermodynamic driving force toward folding. Our study, thus, not only provides experimental evidence and theoretical support for small molecule crowding but also predicts further enhancement of crowding effects for even smaller molecules on a per volume basis.

Eukaryote specific RNA and protein features facilitate assembly and catalysis of H/ACA snoRNPs.

H/ACA Box ribonucleoprotein complexes (RNPs) play a major role in modification of rRNA and snRNA, catalyzing the sequence specific pseudouridylation in eukaryotes and archaea. This enzymatic reaction takes place on a substrate RNA recruited via base pairing to an internal loop of the snoRNA. Eukaryotic snoRNPs contain the four proteins Nop10, Cbf5, Gar1 and Nhp2, with Cbf5 as the catalytic subunit. In contrast to archaeal H/ACA RNPs, eukaryotic snoRNPs contain several conserved features in both the snoRNA as well as the protein components. Here, we reconstituted the eukaryotic H/ACA RNP containing snR81 as a guide RNA in vitro and report on the effects of these eukaryote specific features on complex assembly and enzymatic activity. We compare their contribution to pseudouridylation activity for stand-alone hairpins versus the bipartite RNP. Using single molecule FRET spectroscopy, we investigated the role of the different eukaryote-specific proteins and domains on RNA folding and complex assembly, and assessed binding of substrate RNA to the RNP. Interestingly, we found diverging effects for the two hairpins of snR81, suggesting hairpin-specific requirements for folding and RNP formation. Our results for the first time allow assessing interactions between the individual hairpin RNPs in the context of the full, bipartite snoRNP.

Entropic Inhibition: How the Activity of a AAA+ Machine Is Modulated by Its Substrate-Binding Domain.

ClpB is a tightly regulated AAA+ disaggregation machine. Each ClpB molecule is composed of a flexibly attached N-terminal domain (NTD), an essential middle domain (MD) that activates the machine by tilting, and two nucleotide-binding domains. The NTD is not well-characterized structurally and is commonly considered to serve as a dispensable substrate-binding domain. Here, we use single-molecule FRET spectroscopy to directly monitor the real-time dynamics of ClpB’s NTD and reveal its unexpected autoinhibitory function. We find that the NTD fluctuates on the microsecond time scale, and these dynamics result in steric hindrance that limits the conformational space of the MD to restrict its tilting. This leads to significantly inhibited ATPase and disaggregation activities of ClpB, an effect that is alleviated upon binding of a substrate protein or the cochaperone DnaK. This entropic inhibition mechanism, which is mediated by ultrafast motions of the NTD and is not dependent on any strong interactions, might be common in related ATP-dependent proteases and other multidomain proteins to ensure their fast and reversible activation.

Understanding Microsecond Dynamics of Protein Machines

Protein machines carry out specific tasks in the cell by alternating chemical steps with conformational transitions. Single-molecule FRET spectroscopy is a powerful tool for exposing large-scale function-related motions. We recently developed a novel maximum likelihood algorithm for the analysis of single-molecule experiments, which can track conformational dynamics even on the microsecond time scale (Pirchi et al., JPC B 2016, 120, 13065). We used this algorithm to study two protein machines, the abundant enzyme adenylate kinase (AK) and the disaggregation machine ClpB, finding in both cases surprisingly fast motions.

Untangling the interaction of α-synuclein with DNA i-motifs and hairpins by volume-sensitive single-molecule FRET spectroscopy.

The intrinsically disordered protein α-synuclein causes Parkinson's disease by forming toxic oligomeric aggregates inside neurons. Single-molecule FRET experiments revealed conformational changes of noncanonical DNA structures, such as i-motifs and hairpins, in the presence of α-synuclein. Volumetric analyses revealed differences in binding mode, which is also affected by cellular osmolytes.