RESUMO
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.
Assuntos
Trifosfato de Adenosina , Transferência Ressonante de Energia de Fluorescência , Humanos , Trifosfato de Adenosina/metabolismo , Modelos Moleculares , Proteínas de Choque Térmico/metabolismo , Domínios Proteicos , Adenosina Trifosfatases/metabolismoRESUMO
The chaperonin GroEL is a multisubunit molecular machine that assists in protein folding in the Escherichia coli cytosol. Past studies have shown that GroEL undergoes large allosteric conformational changes during its reaction cycle. Here, we report single-molecule Förster resonance energy transfer measurements that directly probe the conformational transitions of one subunit within GroEL and its single-ring variant under equilibrium conditions. We find that four microstates span the conformational manifold of the protein and interconvert on the submillisecond time scale. A unique set of relative populations of these microstates, termed a macrostate, is obtained by varying solution conditions, e.g., adding different nucleotides or the cochaperone GroES. Strikingly, ATP titration studies demonstrate that the partition between the apo and ATP-ligated conformational macrostates traces a sigmoidal response with a Hill coefficient similar to that obtained in bulk experiments of ATP hydrolysis. These coinciding results from bulk measurements for an entire ring and single-molecule measurements for a single subunit provide new evidence for the concerted allosteric transition of all seven subunits.
Assuntos
Trifosfato de Adenosina , Transferência Ressonante de Energia de Fluorescência , Trifosfato de Adenosina/metabolismo , Conformação Proteica , Escherichia coli/metabolismo , Dobramento de Proteína , Chaperonina 60/metabolismo , Ligação ProteicaRESUMO
Internal motions in proteins take place on a broad range of time- and space-scales. The potential roles of these dynamics in the biochemical functions of proteins have intrigued biophysicists for many years, and multiple mechanisms to couple motions to function have been proposed. Some of these mechanisms have relied on equilibrium concepts. For example, the modulation of dynamics was proposed to change the entropy of a protein, hence affecting processes such as binding. This so-called dynamic allostery scenario has been demonstrated in several recent experiments. Perhaps even more intriguing may be models that involve out-of-equilibrium operation, which by necessity require the input of energy. We discuss several recent experimental studies that expose such potential mechanisms for coupling dynamics and function. In Brownian ratchets, for example, directional motion is promoted by switching a protein between two free energy surfaces. An additional example involves the effect of microsecond domain-closure dynamics of an enzyme on its much slower chemical cycle. These observations lead us to propose a novel two-time-scale paradigm for the activity of protein machines: fast equilibrium fluctuations take place on the microsecond-millisecond time scale, while on a slower time scale, free energy is invested in order to push the system out of equilibrium and drive functional transitions. Motions on the two time scales affect each other and are essential for the overall function of these machines.
Assuntos
Proteínas , Conformação ProteicaRESUMO
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.
Assuntos
Trifosfato de Adenosina , Adenilato Quinase , Adenilato Quinase/metabolismo , Ligantes , Sítios de Ligação , Domínios Proteicos , Trifosfato de Adenosina/metabolismoRESUMO
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.
Assuntos
Proteínas de Escherichia coli , Proteínas de Choque Térmico , Proteínas de Choque Térmico/metabolismo , Endopeptidase Clp/metabolismo , Transferência Ressonante de Energia de Fluorescência , Chaperonas Moleculares/metabolismo , Análise Espectral , Proteínas de Escherichia coli/metabolismo , Trifosfato de Adenosina/metabolismoRESUMO
Considerable electric fields are present within living cells, and the role of bioelectricity has been well established at the organismal level. Yet much remains to be learned about electric-field effects on protein function. Here, we use phototriggered charge injection from a site-specifically attached ruthenium photosensitizer to directly demonstrate the effect of dynamic charge redistribution within a protein. We find that binding of an antibody to phosphoglycerate kinase (PGK) is increased twofold under illumination. Remarkably, illumination is found to suppress the enzymatic activity of PGK by a factor as large as three. These responses are sensitive to the photosensitizer position on the protein. Surprisingly, left (but not right) circularly polarized light elicits these responses, indicating that the electrons involved in the observed dynamics are spin polarized, due to spin filtration by protein chiral structures. Our results directly establish the contribution of electrical polarization as an allosteric signal within proteins. Future experiments with phototriggered charge injection will allow delineation of charge rearrangement pathways within proteins and will further depict their effects on protein function.
Assuntos
Campos Eletromagnéticos , Proteínas , Regulação Alostérica , Elétrons , Iluminação , Fármacos Fotossensibilizantes/farmacologia , Ligação Proteica , Proteínas/efeitos dos fármacos , Proteínas/metabolismo , Proteínas/efeitos da radiação , Rutênio/farmacologiaRESUMO
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.
RESUMO
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.
Assuntos
Endopeptidase Clp/química , Adenosina Trifosfatases/antagonistas & inibidores , Transferência Ressonante de Energia de Fluorescência , Conformação Proteica , Especificidade por SubstratoRESUMO
Protein function may be modulated by an event occurring far away from the functional site, a phenomenon termed allostery. While classically allostery involves conformational changes, we recently observed that charge redistribution within an antibody can also lead to an allosteric effect, modulating the kinetics of binding to target antigen. In the present work, we study the association of a polyhistidine tagged enzyme (phosphoglycerate kinase, PGK) to surface-immobilized anti-His antibodies, finding a significant Charge-Reorganization Allostery (CRA) effect. We further observe that PGK's negatively charged nucleotide substrates modulate CRA substantially, even though they bind far away from the His-tag-antibody interaction interface. In particular, binding of ATP reduces CRA by more than 50%. The results indicate that CRA is affected by the binding of charged molecules to a protein and provide further insight into the significant role that charge redistribution can play in protein function.
Assuntos
Fosfoglicerato Quinase/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Anticorpos/imunologia , Reações Antígeno-Anticorpo , Histidina/genética , Histidina/imunologia , Histidina/metabolismo , Oligopeptídeos/genética , Oligopeptídeos/imunologia , Oligopeptídeos/metabolismo , Fosfoglicerato Quinase/química , Fosfoglicerato Quinase/genética , Conformação Proteica , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por SubstratoRESUMO
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.
Assuntos
Anticorpos Imobilizados/química , Endopeptidase Clp/química , Regulação Alostérica , Sítio Alostérico , Aminoácidos/química , Escherichia coli/genética , Ouro/química , Cinética , Modelos Moleculares , Ligação Proteica , Compostos de Sulfidrila/química , Propriedades de Superfície , Thermus thermophilus/genéticaRESUMO
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.
Assuntos
Endopeptidase Clp/metabolismo , Agregados Proteicos , Thermus thermophilus/enzimologia , Regulação Alostérica , Sítios de Ligação , Endopeptidase Clp/química , Transferência Ressonante de Energia de Fluorescência , Proteínas de Choque Térmico HSP70/metabolismo , Modelos Moleculares , Domínios Proteicos , Especificidade por Substrato , Fatores de TempoRESUMO
Folding of proteins to their functional conformation is paramount to life. Though 75% of the proteome consists of multidomain proteins, our knowledge of folding has been based primarily on studies conducted on single-domain and fast-folding proteins. Nonetheless, the complexity of folding landscapes exhibited by multidomain proteins has received increased scrutiny in recent years. We study the three-domain protein adenylate kinase from E. coli (AK), which has been shown to fold through a series of pathways involving several intermediate states. We use a protein design method to manipulate the folding landscape of AK, and single-molecule FRET spectroscopy to study the effects on the folding process. Mutations introduced in the NMP binding (NMPbind) domain of the protein are found to have unexpected effects on the folding landscape. Thus, while stabilizing mutations in the core of the NMPbind domain retain the main folding pathways of wild-type AK, a destabilizing mutation at the interface between the NMPbind and the CORE domains causes a significant repartition of the flux between the folding pathways. Our results demonstrate the outstanding plasticity of the folding landscape of AK and reveal how specific mutations in the primary structure are translated into changes in folding dynamics. The combination of methodologies introduced in this work should prove useful for deepening our understanding of the folding process of multidomain proteins.
Assuntos
Adenilato Quinase/química , Escherichia coli/enzimologia , Dobramento de Proteína , Adenilato Quinase/genética , Adenilato Quinase/metabolismo , Transferência Ressonante de Energia de Fluorescência , Mutação , Engenharia de ProteínasRESUMO
Experimental tools of increasing sophistication have been employed in recent years to study protein folding and misfolding. Folding is considered a complex process, and one way to address it is by studying small proteins, which seemingly possess a simple energy landscape with essentially only two stable states, either folded or unfolded. The B1-IgG binding domain of protein L (PL) is considered a model two-state folder, based on measurements using a wide range of experimental techniques. We applied single-molecule fluorescence resonance energy transfer (FRET) spectroscopy in conjunction with a hidden Markov model analysis to fully characterize the energy landscape of PL and to extract the kinetic properties of individual molecules of the protein. Surprisingly, our studies revealed the existence of a third state, hidden under the two-state behavior of PL due to its small population, â¼7%. We propose that this minority intermediate involves partial unfolding of the two C-terminal ß strands of PL. Our work demonstrates that single-molecule FRET spectroscopy can be a powerful tool for a comprehensive description of the folding dynamics of proteins, capable of detecting and characterizing relatively rare metastable states that are difficult to observe in ensemble studies.
Assuntos
Modelos Biológicos , Proteínas/química , Transferência Ressonante de Energia de Fluorescência , Simulação de Dinâmica Molecular , Dobramento de ProteínaRESUMO
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.
Assuntos
Adenilato Quinase/química , Adenilato Quinase/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Adenilato Quinase/genética , Sítios de Ligação , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Mutação Puntual , Conformação Proteica , Domínios ProteicosRESUMO
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.
Assuntos
Adenilato Quinase/metabolismo , Proteínas de Escherichia coli/metabolismo , Ligantes , Adenilato Quinase/química , Adenilato Quinase/genética , Adenilil Imidodifosfato/química , Adenilil Imidodifosfato/metabolismo , Dicroísmo Circular , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Escherichia coli/enzimologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Transferência Ressonante de Energia de Fluorescência , Cinética , Ligação Proteica , Desnaturação Proteica , Dobramento de Proteína , Estabilidade Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Especificidade por Substrato , TermodinâmicaRESUMO
Proteins play a crucial role in all living organisms, with the 20 natural amino acids as their building blocks. Unnatural amino acids are synthetic derivatives of these natural building blocks. These amino acids have unique chemical or physical properties as a result of their specific side chain residues. Their incorporation into proteins through ribosomal translation in response to one of the stop codons has opened a new way to manipulate and study proteins by enabling new functionalities, thus expending the genetic code. Different unnatural amino acids have different functionalities, hence, the ability to incorporate two different unnatural amino acids, in response to two different stop codons into one protein is a useful tool in protein manipulation. This ability has been achieved previously only in in vivo translational systems, however, with limited functionality. Herein, we report the incorporation of two different unnatural amino acids in response to two different stop codons into one protein, utilizing a cell-free protein synthesis system. Biotechnol. Bioeng. 2017;114: 1065-1073. © 2016 Wiley Periodicals, Inc.
Assuntos
Aminoácidos/metabolismo , Sistema Livre de Células/metabolismo , Códon de Terminação/metabolismo , Biossíntese de Proteínas , Engenharia de Proteínas/métodos , Aminoácidos/química , Extratos Celulares , Escherichia coli , Transferência Ressonante de Energia de FluorescênciaRESUMO
Leukocyte microvilli are flexible projections enriched with adhesion molecules. The role of these cellular projections in the ability of T cells to probe antigen-presenting cells has been elusive. In this study, we probe the spatial relation of microvilli and T-cell receptors (TCRs), the major molecules responsible for antigen recognition on the T-cell membrane. To this end, an effective and robust methodology for mapping membrane protein distribution in relation to the 3D surface structure of cells is introduced, based on two complementary superresolution microscopies. Strikingly, TCRs are found to be highly localized on microvilli, in both peripheral blood human T cells and differentiated effector T cells, and are barely found on the cell body. This is a decisive demonstration that different types of T cells universally localize their TCRs to microvilli, immediately pointing to these surface projections as effective sensors for antigenic moieties. This finding also suggests how previously reported membrane clusters might form, with microvilli serving as anchors for specific T-cell surface molecules.
Assuntos
Microscopia/métodos , Microvilosidades/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Antígenos CD/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Diferenciação Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Humanos , Imageamento Tridimensional , Selectina L/metabolismo , Linfonodos/citologia , Linfonodos/metabolismo , Proteínas de Membrana/metabolismo , Microvilosidades/efeitos dos fármacos , Microvilosidades/ultraestrutura , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Linfócitos T/ultraestrutura , Tiazolidinas/farmacologiaRESUMO
In contrast to globular proteins, the structure of repeat proteins is dominated by a regular set of short-range interactions. This property may confer on the native state of such proteins significant elasticity. We probe the molecular origin of the spring-like behavior of repeat proteins using a designed tetratricopeptide repeat protein with three repeat units (CTPR3). Single-molecule fluorescence studies of variants of the protein with FRET pairs at different positions show a continuous expansion of the folded state of CTPR3 at low concentrations of a chemical denaturant, preceding the all-or-none transition to the unfolded state. This remarkable native-state expansion can be explained quantitatively by a reduction in the spring constant of the structure. Circular dichroism and tryptophan fluorescence spectroscopy further show that the expansion does not involve either unwinding of CTPR3 helices or unraveling of interactions within repeats. These findings point to hydrophobic inter-repeat contacts as the source of the elasticity of repeat proteins.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas/química , Dicroísmo Circular , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Proteínas/metabolismo , Espectrometria de Fluorescência , Triptofano/químicaRESUMO
Proteins attain their function only after folding into a highly organized three-dimensional structure. Much remains to be learned about the mechanisms of folding of large multidomain proteins, which may populate metastable intermediate states on their energy landscapes. Here we introduce a novel method, based on high-throughput single-molecule fluorescence experiments, which is specifically geared towards tracing the dynamics of folding in the presence of a plethora of intermediates. We employ this method to characterize the folding reaction of a three-domain protein, adenylate kinase. Using thousands of single-molecule trajectories and hidden Markov modelling, we identify six metastable states on adenylate kinase's folding landscape. Remarkably, the connectivity of the intermediates depends on denaturant concentration; at low concentration, multiple intersecting folding pathways co-exist. We anticipate that the methodology introduced here will find broad applicability in the study of folding of large proteins, and will provide a more realistic scenario of their conformational dynamics.
Assuntos
Proteínas/química , Espectrometria de Fluorescência/métodos , Algoritmos , Cadeias de Markov , Dobramento de ProteínaRESUMO
Presynaptic inhibition via G-protein-coupled receptors (GPCRs) and voltage-gated Ca(2+) channels constitutes a widespread regulatory mechanism of synaptic strength. Yet, the mechanism of intermolecular coupling underlying GPCR-mediated signaling at central synapses remains unresolved. Using FRET spectroscopy, we provide evidence for formation of spatially restricted (<100 Å) complexes between GABA(B) receptors composed of GB(1a)/GB(2) subunits, Gα(o)ß(1)γ(2) G-protein heterotrimer, and Ca(V)2.2 channels in hippocampal boutons. GABA release was not required for the assembly but for structural reorganization of the precoupled complex. Unexpectedly, GB(1a) deletion disrupted intermolecular associations within the complex. The GB(1a) proximal C-terminal domain was essential for association of the receptor, Ca(V)2.2 and Gßγ, but was dispensable for agonist-induced receptor activation and cAMP inhibition. Functionally, boutons lacking this complex-formation domain displayed impaired presynaptic inhibition of Ca(2+) transients and synaptic vesicle release. Thus, compartmentalization of the GABA(B1a) receptor, Gßγ, and Ca(V)2.2 channel in a signaling complex is required for presynaptic inhibition at hippocampal synapses.