ABSTRACT
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.
Subject(s)
Adenosine Triphosphate , Fluorescence Resonance Energy Transfer , Humans , Adenosine Triphosphate/metabolism , Models, Molecular , Heat-Shock Proteins/metabolism , Protein Domains , Adenosine Triphosphatases/metabolismABSTRACT
Fluorescence microscopy has witnessed many clever innovations in the last two decades, leading to new methods such as structured illumination and super-resolution microscopies. The attainable resolution in biological samples is, however, ultimately limited by residual motion within the sample or in the microscope setup. Thus, such experiments are typically performed on chemically fixed samples. Cryogenic light microscopy (Cryo-LM) has been investigated as an alternative, drawing on various preservation techniques developed for cryogenic electron microscopy (Cryo-EM). Moreover, this approach offers a powerful platform for correlative microscopy. Another key advantage of Cryo-LM is the strong reduction in photobleaching at low temperatures, facilitating the collection of orders of magnitude more photons from a single fluorophore. This results in much higher localization precision, leading to Angstrom resolution. In this review, we discuss the general development and progress of Cryo-LM with an emphasis on its application in harnessing structural information on proteins and protein complexes.
Subject(s)
Cold Temperature , Cryoelectron Microscopy/methods , Microscopy, Fluorescence/methods , Microscopy, ElectronABSTRACT
Interferometric scattering (iSCAT) microscopy is a label-free optical method capable of detecting single proteins, localizing their binding positions with nanometer precision, and measuring their mass. In the ideal case, iSCAT is limited by shot noise such that collection of more photons should extend its detection sensitivity to biomolecules of arbitrarily low mass. However, a number of technical noise sources combined with speckle-like background fluctuations have restricted the detection limit in iSCAT. Here, we show that an unsupervised machine learning isolation forest algorithm for anomaly detection pushes the mass sensitivity limit by a factor of 4 to below 10 kDa. We implement this scheme both with a user-defined feature matrix and a self-supervised FastDVDNet and validate our results with correlative fluorescence images recorded in total internal reflection mode. Our work opens the door to optical investigations of small traces of biomolecules and disease markers such as α-synuclein, chemokines and cytokines.
Subject(s)
Microscopy , Photons , Cytokines , Supervised Machine LearningABSTRACT
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.
Subject(s)
Escherichia coli Proteins , Heat-Shock Proteins , Heat-Shock Proteins/metabolism , Endopeptidase Clp/metabolism , Fluorescence Resonance Energy Transfer , Molecular Chaperones/metabolism , Spectrum Analysis , Escherichia coli Proteins/metabolism , Adenosine Triphosphate/metabolismABSTRACT
Cryogenic optical localization in three dimensions (COLD) was recently shown to resolve up to four binding sites on a single protein. However, because COLD relies on intensity fluctuations that result from the blinking behavior of fluorophores, it is limited to cases where individual emitters show different brightness. This significantly lowers the measurement yield. To extend the number of resolved sites as well as the measurement yield, we employ partial labeling and combine it with polarization encoding in order to identify single fluorophores during their stochastic blinking. We then use a particle classification scheme to identify and resolve heterogenous subsets and combine them to reconstruct the three-dimensional arrangement of large molecular complexes. We showcase this method (polarCOLD) by resolving the trimer arrangement of proliferating cell nuclear antigen (PCNA) and six different sites of the hexamer protein Caseinolytic Peptidase B (ClpB) of Thermus thermophilus in its quaternary structure, both with Angstrom resolution. The combination of polarCOLD and single-particle cryogenic electron microscopy (cryoEM) promises to provide crucial insight into intrinsic heterogeneities of biomolecular structures. Furthermore, our approach is fully compatible with fluorescent protein labeling and can, thus, be used in a wide range of studies in cell and membrane biology.
Subject(s)
Fluorescent Dyes , Single Molecule Imaging , Microscopy, Fluorescence/methods , Single Molecule Imaging/methodsABSTRACT
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.
ABSTRACT
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.
Subject(s)
Endopeptidase Clp/chemistry , Adenosine Triphosphatases/antagonists & inhibitors , Fluorescence Resonance Energy Transfer , Protein Conformation , Substrate SpecificityABSTRACT
A new mechanism of allostery in proteins, based on charge rather than structure, is reported. We demonstrate that dynamic redistribution of charge within a protein can control its function and affect its interaction with a binding partner. In particular, the association of an antibody with its target protein antigen is studied. Dynamic charge shifting within the antibody during its interaction with the antigen is enabled by its binding to a metallic surface that serves as a source for electrons. The kinetics of antibody-antigen association are enhanced when charge redistribution is allowed, even though charge injection happens at a position far from the antigen binding site. This observation points to charge-reorganization allostery, which should be operative in addition or parallel to other mechanisms of allostery, and may explain some current observations on protein interactions.
Subject(s)
Antibodies, Immobilized/chemistry , Endopeptidase Clp/chemistry , Allosteric Regulation , Allosteric Site , Amino Acids/chemistry , Escherichia coli/genetics , Gold/chemistry , Kinetics , Models, Molecular , Protein Binding , Sulfhydryl Compounds/chemistry , Surface Properties , Thermus thermophilus/geneticsABSTRACT
Protein motions occur on multiple time and distance scales. Large-scale motions of protein tertiary-structure elements, i.e., domains, are particularly intriguing as they are essential for the catalytic activity of many enzymes and for the functional cycles of protein machines and motors. Theoretical estimates suggest that domain motions should be very fast, occurring on the nanosecond or microsecond time scales. Indeed, free-energy barriers for domain motions are likely to involve salt bridges, which can break in microseconds. Experimental methods that can directly probe domain motions on fast time scales have appeared only in recent years. This Perspective discusses briefly some of these techniques, including nuclear magnetic resonance and single-molecule fluorescence spectroscopies. We introduce a few recent studies that demonstrate ultrafast domain motions and discuss their potential roles. Particularly surprising is the observation of tertiary-structure element dynamics that are much faster than the functional cycles in some protein machines. These swift motions can be rationalized on a case-by-case basis. For example, fast domain closure in multi-substrate enzymes may be utilized to optimize relative substrate orientation. Whether a large mismatch in time scales of conformational dynamics vs functional cycles is a general design principle in proteins remains to be determined.
Subject(s)
Motion , Protein Domains , Proteins/chemistry , Fluorescence Resonance Energy Transfer , Molecular Dynamics Simulation , Nuclear Magnetic Resonance, BiomolecularABSTRACT
The thermodynamics of protein folding in bulk solution have been thoroughly investigated for decades. By contrast, measurements of protein substrate stability inside the GroEL/ES chaperonin cage have not been reported. Such measurements require stable encapsulation, that is no escape of the substrate into bulk solution during experiments, and a way to perturb protein stability without affecting the chaperonin system itself. Here, by establishing such conditions, we show that protein stability in the chaperonin cage is reduced dramatically by more than 5 kcal mol-1 compared to that in bulk solution. Given that steric confinement alone is stabilizing, our results indicate that hydrophobic and/or electrostatic effects in the cavity are strongly destabilizing. Our findings are consistent with the iterative annealing mechanism of action proposed for the chaperonin GroEL.
All cells contain molecules known as proteins that perform many essential roles. Proteins are made of chains of building blocks called amino acids that fold to form the proteins' three-dimensional structures. Many proteins fold spontaneously into their well-defined and correct structures. However, some proteins fold incorrectly, which prevents them from working properly, and can lead to formation of aggregates that may harm the cell. To prevent such damage, cells have evolved proteins known as molecular chaperones that assist in the folding of other proteins. For example, a molecular chaperone called GroEL is found in a bacterium known as Escherichia coli. This molecular chaperone contains a cavity which prevents target proteins from forming clumps by keeping them away from other proteins. However, it remained unclear precisely how GroEL works and whether enclosing target proteins in its cavity has other effects. Moritella profunda is a bacterium that thrives in cold environments and, as a result, many of its proteins are unstable at room temperature and tend to unfold or fold incorrectly. To study how GroEL works, Korobko et al. used a protein from M. profunda called dihydrofolate reductase as a target protein for the chaperone. A clever trick was then used to determine the folding state of dihydrofolate reductase when inside the chaperone cavity. The experiments revealed that the environment within the cavity of GroEL strongly favors dihydrofolate reductase adopting its unfolded state instead of its folded state. This suggests that GroEL helps dihydrofolate reductase and other incorrectly folded target proteins to unfold, thus providing the proteins another opportunity to fold again correctly. Parkinson's disease, Alzheimer's disease and many other diseases are caused by proteins folding incorrectly and forming aggregates. A better understanding of how proteins fold may, therefore, assist in developing new therapies for such diseases. These findings may also help biotechnology researchers develop methods for producing difficult-to-fold proteins on a large scale.
Subject(s)
Chaperonins/metabolism , Escherichia coli Proteins/metabolism , Heat-Shock Proteins/metabolism , Protein Folding , Tetrahydrofolate Dehydrogenase/metabolism , Chaperonins/chemistry , Escherichia coli/metabolism , Escherichia coli Proteins/chemistry , Heat-Shock Proteins/chemistry , Hydrophobic and Hydrophilic Interactions , Moritella/metabolism , Protein Aggregation, PathologicalABSTRACT
Hsp90 plays a central role in cell homeostasis by assisting folding and maturation of a large variety of clients. It is a homo-dimer, which functions via hydrolysis of ATP-coupled to conformational changes. Hsp90's conformational cycle in the absence of cochaperones is currently postulated as apo-Hsp90 being an ensemble of "open"/"closed" conformations. Upon ATP binding, Hsp90 adopts an active ATP-bound closed conformation where the N-terminal domains, which comprise the ATP binding site, are in close contact. However, there is no consensus regarding the conformation of the ADP-bound Hsp90, which is considered important for client release. In this work, we tracked the conformational states of yeast Hsp90 at various stages of ATP hydrolysis in frozen solutions employing electron paramagnetic resonance (EPR) techniques, particularly double electron-electron resonance (DEER) distance measurements. Using rigid Gd(III) spin labels, we found the C domains to be dimerized with same distance distribution at all hydrolysis states. Then, we substituted the ATPase Mg(II) cofactor with paramagnetic Mn(II) and followed the hydrolysis state using hyperfine spectroscopy and measured the inter-N-domain distance distributions via Mn(II)-Mn(II) DEER. The point character of the Mn(II) spin label allowed us resolve 2 different closed states: The ATP-bound (prehydrolysis) characterized by a distance distribution having a maximum of 4.3 nm, which broadened and shortened, shifting the mean to 3.8 nm at the ADP-bound state (posthydrolysis). This provides experimental evidence to a second closed conformational state of Hsp90 in solution, referred to as "compact." Finally, the so-called high-energy state, trapped by addition of vanadate, was found structurally similar to the posthydrolysis state.
Subject(s)
Fungal Proteins/metabolism , HSP90 Heat-Shock Proteins/metabolism , Protein Domains/genetics , Yeasts/metabolism , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Electron Spin Resonance Spectroscopy , Fungal Proteins/chemistry , Fungal Proteins/genetics , HSP90 Heat-Shock Proteins/chemistry , HSP90 Heat-Shock Proteins/genetics , Manganese/chemistry , Models, Molecular , Mutation , Spin Labels , Yeasts/geneticsABSTRACT
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.
Subject(s)
Endopeptidase Clp/metabolism , Protein Aggregates , Thermus thermophilus/enzymology , Allosteric Regulation , Binding Sites , Endopeptidase Clp/chemistry , Fluorescence Resonance Energy Transfer , HSP70 Heat-Shock Proteins/metabolism , Models, Molecular , Protein Domains , Substrate Specificity , Time FactorsABSTRACT
Feynman commented that "Everything that living things do can be understood in terms of the jiggling and wiggling of atoms". Proteins can jiggle and wiggle large structural elements such as domains and subunits as part of their functional cycles. Single-molecule fluorescence resonance energy transfer (smFRET) is an excellent tool to study conformational dynamics and decipher coordinated large-scale motions within proteins. smFRET methods introduced in recent years are geared toward understanding the time scales and amplitudes of function-related motions. This review discusses the methodology for obtaining and analyzing smFRET temporal trajectories that provide direct dynamic information on transitions between conformational states. It also introduces correlation methods that are useful for characterizing intramolecular motions. This arsenal of techniques has been used to study multiple molecular systems, from membrane proteins through molecular chaperones, and we examine some of these studies here. Recent exciting methodological novelties permit revealing very fast, submillisecond dynamics, whose relevance to protein function is yet to be fully grasped.
ABSTRACT
The functional cycle of many proteins involves large-scale motions of domains and subunits. The relation between conformational dynamics and the chemical steps of enzymes remains under debate. Here we show that in the presence of substrates, domain motions of an enzyme can take place on the microsecond time scale, yet exert influence on the much-slower chemical step. We study the domain closure reaction of the enzyme adenylate kinase from Escherichia coli while in action (i.e., under turnover conditions), using single-molecule FRET spectroscopy. We find that substrate binding increases dramatically domain closing and opening times, making them as short as â¼15 and â¼45 µs, respectively. These large-scale conformational dynamics are likely the fastest measured to date, and are â¼100-200 times faster than the enzymatic turnover rate. Some active-site mutants are shown to fully or partially prevent the substrate-induced increase in domain closure times, while at the same time they also reduce enzymatic activity, establishing a clear connection between the two phenomena, despite their disparate time scales. Based on these surprising observations, we propose a paradigm for the mode of action of enzymes, in which numerous cycles of conformational rearrangement are required to find a mutual orientation of substrates that is optimal for the chemical reaction.
Subject(s)
Adenylate Kinase/chemistry , Adenylate Kinase/metabolism , Fluorescence Resonance Energy Transfer/methods , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Adenylate Kinase/genetics , Binding Sites , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Point Mutation , Protein Conformation , Protein DomainsABSTRACT
Ligand binding to a protein can stabilize it significantly against unfolding. The variation of the folding free energy, ΔΔG0, due to ligand binding can be derived from a simple reaction scheme involving exclusive binding to the native state. One obtains the following expression: , where Kd is the ligand dissociation constant and L is its concentration, R is the universal gas constant and T is the temperature. This expression has been shown to correctly describe experimental results on multiple proteins. In the current work we studied the effect of ligand binding on the stability of the multi-domain protein adenylate kinase from E. coli (AKE). Unfolding experiments were conducted using single-molecule FRET spectroscopy, which allowed us to directly obtain the fraction of unfolded protein in a model-free way from FRET efficiency histograms. Surprisingly, it was found that the effect of two inhibitors (Ap5A and AMPPNP) and a substrate (AMP) on the stability of AKE was much smaller than expected based on Kd values obtained independently using microscale thermophoresis. To shed light on this issue, we measured the Kd for Ap5A over a range of chemical denaturant concentrations where the protein is still folded. It was found that Kd increases dramatically over this range, likely due to the population of folding intermediates, whose binding to the ligand is much weaker than that of the native state. We propose that binding to folding intermediates may dominate the effect of ligands on the stability of multi-domain proteins, and could therefore have a strong impact on protein homeostasis in vivo.