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.

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.

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.

Ultrafast pore-loop dynamics in a AAA+ machine point to a Brownian-ratchet mechanism for protein translocation.

AAA+ ring­shaped machines, such as the disaggregation machines ClpB and Hsp104, mediate ATP-driven substrate translocation through their central channel by a set of pore loops. Recent structural studies have suggested a universal hand-over-hand translocation mechanism with slow and rigid subunit motions. However, functional and biophysical studies are in discord with this model. Here, we directly measure the real-time dynamics of the pore loops of ClpB during substrate threading, using single-molecule FRET spectroscopy. All pore loops undergo large-amplitude fluctuations on the microsecond time scale and change their conformation upon interaction with substrate proteins in an ATP-dependent manner. Conformational dynamics of two of the pore loops strongly correlate with disaggregation activity, suggesting that they are the main contributors to substrate pulling. This set of findings is rationalized in terms of an ultrafast Brownian-ratchet translocation mechanism, which likely acts in parallel to the much slower hand-over-hand process in ClpB and other AAA+ machines.

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.

Tunable microsecond dynamics of an allosteric switch regulate the activity of a AAA+ disaggregation machine.

Large protein machines are tightly regulated through allosteric communication channels. Here we demonstrate the involvement of ultrafast conformational dynamics in allosteric regulation of ClpB, a hexameric AAA+ machine that rescues aggregated proteins. Each subunit of ClpB contains a unique coiled-coil structure, the middle domain (M domain), proposed as a control element that binds the co-chaperone DnaK. Using single-molecule FRET spectroscopy, we probe the M domain during the chaperone cycle and find it to jump on the microsecond time scale between two states, whose structures are determined. The M-domain jumps are much faster than the overall activity of ClpB, making it an effectively continuous, tunable switch. Indeed, a series of allosteric interactions are found to modulate the dynamics, including binding of nucleotides, DnaK and protein substrates. This mode of dynamic control enables fast cellular adaptation and may be a general mechanism for the regulation of cellular machineries.

Quantifying Co-Oligomer Formation by α-Synuclein.

Small oligomers of the protein α-synuclein (αS) are highly cytotoxic species associated with Parkinson's disease (PD). In addition, αS can form co-aggregates with its mutational variants and with other proteins such as amyloid-ß (Aß) and tau, which are implicated in Alzheimer's disease. The processes of self-oligomerization and co-oligomerization of αS are, however, challenging to study quantitatively. Here, we have utilized single-molecule techniques to measure the equilibrium populations of oligomers formed in vitro by mixtures of wild-type αS with its mutational variants and with Aß40, Aß42, and a fragment of tau. Using a statistical mechanical model, we find that co-oligomer formation is generally more favorable than self-oligomer formation at equilibrium. Furthermore, self-oligomers more potently disrupt lipid membranes than do co-oligomers. However, this difference is sometimes outweighed by the greater formation propensity of co-oligomers when multiple proteins coexist. Our results suggest that co-oligomer formation may be important in PD and related neurodegenerative diseases.

Nanobodies raised against monomeric ɑ-synuclein inhibit fibril formation and destabilize toxic oligomeric species.

BACKGROUND: The aggregation of the protein ɑ-synuclein (ɑS) underlies a range of increasingly common neurodegenerative disorders including Parkinson's disease. One widely explored therapeutic strategy for these conditions is the use of antibodies to target aggregated ɑS, although a detailed molecular-level mechanism of the action of such species remains elusive. Here, we characterize ɑS aggregation in vitro in the presence of two ɑS-specific single-domain antibodies (nanobodies), NbSyn2 and NbSyn87, which bind to the highly accessible C-terminal region of ɑS. RESULTS: We show that both nanobodies inhibit the formation of ɑS fibrils. Furthermore, using single-molecule fluorescence techniques, we demonstrate that nanobody binding promotes a rapid conformational conversion from more stable oligomers to less stable oligomers of ɑS, leading to a dramatic reduction in oligomer-induced cellular toxicity. CONCLUSIONS: The results indicate a novel mechanism by which diseases associated with protein aggregation can be inhibited, and suggest that NbSyn2 and NbSyn87 could have significant therapeutic potential.

Arachidonic acid mediates the formation of abundant alpha-helical multimers of alpha-synuclein.

The protein alpha-synuclein (αS) self-assembles into toxic beta-sheet aggregates in Parkinson's disease, while it is proposed that αS forms soluble alpha-helical multimers in healthy neurons. Here, we have made αS multimers in vitro using arachidonic acid (ARA), one of the most abundant fatty acids in the brain, and characterized them by a combination of bulk experiments and single-molecule FÓ§rster resonance energy transfer (sm-FRET) measurements. The data suggest that ARA-induced oligomers are alpha-helical, resistant to fibril formation, more prone to disaggregation, enzymatic digestion and degradation by the 26S proteasome, and lead to lower neuronal damage and reduced activation of microglia compared to the oligomers formed in the absence of ARA. These multimers can be formed at physiologically-relevant concentrations, and pathological mutants of αS form less multimers than wild-type αS. Our work provides strong biophysical evidence for the formation of alpha-helical multimers of αS in the presence of a biologically relevant fatty acid, which may have a protective role with respect to the generation of beta-sheet toxic structures during αS fibrillation.

Quantitative analysis of co-oligomer formation by amyloid-beta peptide isoforms.

Multiple isoforms of aggregation-prone proteins are present under physiological conditions and have the propensity to assemble into co-oligomers with different properties from self-oligomers, but this process has not been quantitatively studied to date. We have investigated the amyloid-ß (Aß) peptide, associated with Alzheimer's disease, and the aggregation of its two major isoforms, Aß40 and Aß42, using a statistical mechanical modelling approach in combination with in vitro single-molecule fluorescence measurements. We find that at low concentrations of Aß, corresponding to its physiological abundance, there is little free energy penalty in forming co-oligomers, suggesting that the formation of both self-oligomers and co-oligomers is possible under these conditions. Our model is used to predict the oligomer concentration and size at physiological concentrations of Aß and suggests the mechanisms by which the ratio of Aß42 to Aß40 can affect cell toxicity. An increased ratio of Aß42 to Aß40 raises the fraction of oligomers containing Aß42, which can increase the hydrophobicity of the oligomers and thus promote deleterious binding to the cell membrane and increase neuronal damage. Our results suggest that co-oligomers are a common form of aggregate when Aß isoforms are present in solution and may potentially play a significant role in Alzheimer's disease.

Kinetic model of the aggregation of alpha-synuclein provides insights into prion-like spreading.

The protein alpha-synuclein (αS) self-assembles into small oligomeric species and subsequently into amyloid fibrils that accumulate and proliferate during the development of Parkinson's disease. However, the quantitative characterization of the aggregation and spreading of αS remains challenging to achieve. Previously, we identified a conformational conversion step leading from the initially formed oligomers to more compact oligomers preceding fibril formation. Here, by a combination of single-molecule fluorescence measurements and kinetic analysis, we find that the reaction in solution involves two unimolecular structural conversion steps, from the disordered to more compact oligomers and then to fibrils, which can elongate by further monomer addition. We have obtained individual rate constants for these key microscopic steps by applying a global kinetic analysis to both the decrease in the concentration of monomeric protein molecules and the increase in oligomer concentrations over a 0.5-140-µM range of αS. The resulting explicit kinetic model of αS aggregation has been used to quantitatively explore seeding the reaction by either the compact oligomers or fibrils. Our predictions reveal that, although fibrils are more effective at seeding than oligomers, very high numbers of seeds of either type, of the order of 10(4), are required to achieve efficient seeding and bypass the slow generation of aggregates through primary nucleation. Complementary cellular experiments demonstrated that two orders of magnitude lower numbers of oligomers were sufficient to generate high levels of reactive oxygen species, suggesting that effective templated seeding is likely to require both the presence of template aggregates and conditions of cellular stress.

Single-Molecule Imaging of Individual Amyloid Protein Aggregates in Human Biofluids.

The misfolding and aggregation of proteins into amyloid fibrils characterizes many neurodegenerative disorders such as Parkinson's and Alzheimer's diseases. We report here a method, termed SAVE (single aggregate visualization by enhancement) imaging, for the ultrasensitive detection of individual amyloid fibrils and oligomers using single-molecule fluorescence microscopy. We demonstrate that this method is able to detect the presence of amyloid aggregates of α-synuclein, tau, and amyloid-ß. In addition, we show that aggregates can also be identified in human cerebrospinal fluid (CSF). Significantly, we see a twofold increase in the average aggregate concentration in CSF from Parkinson's disease patients compared to age-matched controls. Taken together, we conclude that this method provides an opportunity to characterize the structural nature of amyloid aggregates in a key biofluid, and therefore has the potential to study disease progression in both animal models and humans to enhance our understanding of neurodegenerative disorders.

Fast flow microfluidics and single-molecule fluorescence for the rapid characterization of α-synuclein oligomers.

α-Synuclein oligomers can be toxic to cells and may be responsible for cell death in Parkinson's disease. Their typically low abundance and highly heterogeneous nature, however, make such species challenging to study using traditional biochemical techniques. By combining fast-flow microfluidics with single-molecule fluorescence, we are able to rapidly follow the process by which oligomers of αS are formed and to characterize the species themselves. We have used the technique to show that populations of oligomers with different FRET efficiencies have varying stabilities when diluted into low ionic strength solutions. Interestingly, we have found that oligomers formed early in the aggregation pathway have electrostatic repulsions that are shielded in the high ionic strength buffer and therefore dissociate when diluted into lower ionic strength solutions. This property can be used to isolate different structural groups of αS oligomers and can help to rationalize some aspects of αS amyloid fibril formation.

Inelastic scattering of OH radicals from organic liquids: isolating the thermal desorption channel.

Inelastic scattering of OH radicals from liquid surfaces has been investigated experimentally. An initially translationally and rotationally hot distribution of OH was generated by 193 nm photolysis of allyl alcohol. These radicals were scattered from an inert reference liquid, perfluorinated polyether (PFPE), and from the potentially reactive hydrocarbon liquids squalane (C30H62, 2,6,10,15,19,23-hexamethyltetracosane) and squalene (C30H50, trans-2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene). The scattered OH v = 0 products were detected by laser-induced fluorescence. Strong correlations were observed between the translational and rotational energies of the products. The high-N levels are translationally hot, consistent with a predominantly direct, impulsive scattering mechanism. Impulsive scattering also populates the lower-N levels, but a component of translationally relaxed OH, with thermal-desorption characteristics, can also be seen clearly for all three liquids. More of this translationally and rotationally relaxed OH survives from squalane than from squalene. Realistic molecular dynamics simulations confirm that double-bond sites are accessible at the squalene surface. This supports the proposition that relaxed OH may be lost on squalene via an addition mechanism.

Time-resolved photoelectron imaging of excited state relaxation dynamics in phenol, catechol, resorcinol, and hydroquinone.

Time-resolved photoelectron imaging was used to investigate the dynamical evolution of the initially prepared S(1) (ππ*) excited state of phenol (hydroxybenzene), catechol (1,2-dihydroxybenzene), resorcinol (1,3-dihydroxybenzene), and hydroquinone (1,4-dihydroxybenzene) following excitation at 267 nm. Our analysis was supported by ab initio calculations at the coupled-cluster and CASSCF levels of theory. In all cases, we observe rapid (<1 ps) intramolecular vibrational redistribution on the S(1) potential surface. In catechol, the overall S(1) state lifetime was observed to be 12.1 ps, which is 1-2 orders of magnitude shorter than in the other three molecules studied. This may be attributed to differences in the H atom tunnelling rate under the barrier formed by a conical intersection between the S(1) state and the close lying S(2) (πσ*) state, which is dissociative along the O-H stretching coordinate. Further evidence of this S(1)/S(2) interaction is also seen in the time-dependent anisotropy of the photoelectron angular distributions we have observed. Our data analysis was assisted by a matrix inversion method for processing photoelectron images that is significantly faster than most other previously reported approaches and is extremely quick and easy to implement.