RESUMEN
P-type ATPases are found in all kingdoms of life and constitute a wide range of cation transporters, primarily for H+, Na+, K+, Ca2+, and transition metal ions such as Cu(I), Zn(II), and Cd(II). They have been studied through a wide range of techniques, and research has gained very significant insight on their transport mechanism and regulation. Here, we review the structure, function, and dynamics of P2-ATPases including Ca2+-ATPases and Na,K-ATPase. We highlight mechanisms of functional transitions that are associated with ion exchange on either side of the membrane and how the functional cycle is regulated by interaction partners, autoregulatory domains, and off-cycle states. Finally, we discuss future perspectives based on emerging techniques and insights.
Asunto(s)
Adenosina Trifosfato/química , ATPasas Transportadoras de Cobre/química , ATPasa Intercambiadora de Hidrógeno-Potásio/química , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/química , ATPasa Intercambiadora de Sodio-Potasio/química , Adenosina Trifosfato/metabolismo , Animales , Sitios de Unión , Cationes Bivalentes , Cationes Monovalentes , ATPasas Transportadoras de Cobre/genética , ATPasas Transportadoras de Cobre/metabolismo , ATPasa Intercambiadora de Hidrógeno-Potásio/genética , ATPasa Intercambiadora de Hidrógeno-Potásio/metabolismo , Humanos , Transporte Iónico , Modelos Moleculares , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Protones , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Imagen Individual de Molécula , ATPasa Intercambiadora de Sodio-Potasio/genética , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Especificidad por SustratoRESUMEN
Many kinases use reversible docking interactions to augment the specificity of their catalytic domains. Such docking interactions are often structurally independent of the catalytic domain, which allow for a flexible combination of modules in evolution and in bioengineering. The affinity of docking interactions spans several orders of magnitude. This led us to ask how the affinity of the docking interaction affects enzymatic activity and how to pick the optimal interaction module to complement a given substrate. Here, we develop equations that predict the optimal binding strength of a kinase docking interaction and validate it using numerical simulations and steady-state phosphorylation kinetics for tethered protein kinase A. We show that a kinase-substrate pair has an optimum docking strength that depends on their enzymatic constants, the tether architecture, the substrate concentration, and the kinetics of the docking interactions. We show that a reversible tether enhances phosphorylation rates most when 1) the docking strength is intermediate, 2) the substrate is nonoptimal, 3) the substrate concentration is low, 4) the docking interaction has rapid exchange kinetics, and 5) the tether optimizes the effective concentration of the intramolecular reaction. This work serves as a framework for interpreting mutations in kinase docking interactions and as a design guide for engineering enzyme scaffolds.
Asunto(s)
Dominio Catalítico , Proteínas Quinasas Dependientes de AMP Cíclico , Modelos Químicos , Proteínas Quinasas Dependientes de AMP Cíclico/química , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Guinea Ecuatorial , Cinética , Mutación , Fosforilación , Unión Proteica , Especificidad por SustratoRESUMEN
Phosphorylation-type (P-type) ATPases are ubiquitous primary transporters that pump cations across cell membranes through the formation and breakdown of a phosphoenzyme intermediate. Structural investigations suggest that the transport mechanism is defined by conformational changes in the cytoplasmic domains of the protein that are allosterically coupled to transmembrane helices so as to expose ion binding sites to alternate sides of the membrane. Here, we have used single-molecule fluorescence resonance energy transfer to directly observe conformational changes associated with the functional transitions in the Listeria monocytogenes Ca2+-ATPase (LMCA1), an orthologue of eukaryotic Ca2+-ATPases. We identify key intermediates with no known crystal structures and show that Ca2+ efflux by LMCA1 is rate-limited by phosphoenzyme formation. The transport process involves reversible steps and an irreversible step that follows release of ADP and extracellular release of Ca2+.
Asunto(s)
Adenosina Trifosfato/metabolismo , ATPasas Transportadoras de Calcio/química , ATPasas Transportadoras de Calcio/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Listeria monocytogenes/enzimología , Imagen Individual de Molécula , Adenosina Difosfato/metabolismo , Sitios de Unión , Calcio/metabolismo , Cinética , Modelos Moleculares , Fosforilación , Conformación ProteicaRESUMEN
Kinase specificity is crucial to the fidelity of signaling pathways, yet many pathways use the same kinases to achieve widely different effects. Specificity arises in part from the enzymatic domain but also from the physical tethering of kinases to their substrates. Such tethering can occur via protein interaction domains in the kinase or via anchoring and scaffolding proteins and can drastically increase the kinetics of phosphorylation. However, we do not know how such intracomplex reactions depend on the link between enzyme and substrate. Here we show that the kinetics of tethered kinases follow a Michaelis-Menten-like dependence on effective concentration. We find that phosphorylation kinetics scale with the length of the intrinsically disordered linkers that join the enzyme and substrate but that the scaling differs between substrates. Steady-state kinetics can only partially predict rates of tethered reactions as product release may obscure the rate of phosphotransfer. Our results suggest that changes in signaling complex architecture not only enhance the rates of phosphorylation reactions but may also alter the relative substrate usage. This suggests a mechanism for how scaffolding proteins can allosterically modify the output from a signaling pathway.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Modelos Químicos , Fosforilación , Escherichia coli , CinéticaRESUMEN
Proteins and protein assemblies often tether interaction partners to strengthen interactions, to regulate activity through auto-inhibition or -activation, or to boost enzyme catalysis. Tethered reactions are regulated by the architecture of the tether, which defines an effective concentration of the interactor. Effective concentrations can be estimated theoretically for simple linkers via polymer models, but there is currently no general method for estimating effective concentrations for complex linker architectures consisting of both flexible and folded domains. We describe how effective concentrations can be estimated computationally for any protein linker architecture by defining a realistic conformational ensemble. We benchmark against prediction from a worm-like chain and values measured by competition experiments and find minor differences likely due to excluded volume effects. Systematic variation of the properties of flexible and folded segments shows that the effective concentration is mainly determined by the combination of the total length of flexible segments and the distance between the termini of the folded domains. We show that a folded domain in a disordered linker can increase the effective concentration beyond what can be achieved by a fully disordered linker by focusing the end-to-end distance at the appropriate spacing. This suggests that complex linker architecture may have advantages over simple flexible linkers and emphasizes that annotation as a linker should depend on the molecular context.
Asunto(s)
Dominios Proteicos , Proteínas/química , Catálisis , Enzimas/química , Ligandos , Modelos Moleculares , Conformación Molecular , Peso Molecular , Conformación Proteica , Pliegue de ProteínaRESUMEN
Many multidomain proteins contain disordered linkers that regulate interdomain contacts, and thus the effective concentrations that govern intramolecular reactions. Effective concentrations are rarely measured experimentally, and therefore little is known about how they relate to linker architecture. We have directly measured the effective concentrations enforced by disordered protein linkers using a fluorescent biosensor. We show that effective concentrations follow simple geometric models based on polymer physics, offering an indirect method to probe the structural properties of the linker. The compaction of the disordered linker depends not only on net charge, but also on the type of charged residues. In contrast to theoretical predictions, we found that polyampholyte linkers can contract to similar dimensions as globular proteins. Hydrophobicity has little effect in itself, but aromatic residues lead to strong compaction, likely through π-interactions. Finally, we find that the individual contributors to chain compaction are not additive. We thus demonstrate that direct measurement of effective concentrations can be used in systematic studies of the relationship between sequence and structure of intrinsically disordered proteins. A quantitative understanding of the relationship between effective concentration and linker sequence will be crucial for understanding disorder-based allosteric regulation in multidomain proteins.
Asunto(s)
Proteínas Intrínsecamente Desordenadas/metabolismo , Fenómenos Biofísicos , Dominios ProteicosRESUMEN
NMDA receptors are part of the ionotropic glutamate receptor family, and are crucial for neurotransmission and memory. At the cellular level, the effects of activating these receptors include long-term potentiation (LTP) or depression (LTD). The NMDA receptor is a stringently gated cation channel permeable to Ca2+ , and it shares the molecular architecture of a tetrameric ligand-gated ion channel with the other family members. Its subunits, however, have uniquely long cytoplasmic C-terminal domains (CTDs). While the molecular gymnastics of the extracellular domains have been described in exquisite detail, much less is known about the structure and function of these CTDs. The CTDs vary dramatically in length and sequence between receptor subunits, but they all have a composition characteristic of intrinsically disordered proteins. The CTDs affect channel properties, trafficking and downstream signalling output from the receptor, and these functions are regulated by alternative splicing, protein-protein interactions, and post-translational modifications such as phosphorylation and palmitoylation. Here, we review the roles of the CTDs in synaptic plasticity with a focus on biochemical mechanisms. In total, the CTDs play a multifaceted role as a modifier of channel function, a regulator of cellular location and abundance, and signalling scaffold control the downstream signalling output.
Asunto(s)
Potenciación a Largo Plazo , Receptores de N-Metil-D-Aspartato , Plasticidad Neuronal , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Transducción de Señal , Transmisión SinápticaRESUMEN
Intrinsically disordered proteins organize interaction networks in the cell in many regulation and signaling processes. These proteins often gain structure upon binding to their target proteins in multistep reactions involving the formation of both secondary and tertiary structure. To understand the interactions of disordered proteins, we need to understand the mechanisms of these coupled folding and binding reactions. We studied helix formation in the binding of the molten globule-like nuclear coactivator binding domain and the disordered interaction domain from activator of thyroid hormone and retinoid receptors. We demonstrate that helix formation in a rapid binding reaction can be followed by stopped-flow synchrotron-radiation circular dichroism (CD) spectroscopy and describe the design of such a beamline. Fluorescence-monitored binding experiments of activator of thyroid hormone and retinoid receptors and nuclear coactivator binding domain display several kinetic phases, including one concentration-independent phase, which is consistent with an intermediate stabilized at high ionic strength. Time-resolved CD experiments show that almost all helicity is formed upon initial association of the proteins or separated from the encounter complex by only a small energy barrier. Through simulation of mechanistic models, we show that the intermediate observed at high ionic strength likely involves a structural rearrangement with minor overall changes in helicity. Our experiments provide a benchmark for simulations of coupled binding reactions and demonstrate the feasibility of using synchrotron-radiation CD for mechanistic studies of protein-protein interactions.
Asunto(s)
Dicroismo Circular/métodos , Proteínas Intrínsecamente Desordenadas/química , Pliegue de Proteína , Proteína de Unión a CREB/química , Dicroismo Circular/instrumentación , Humanos , Coactivador 3 de Receptor Nuclear/química , Conformación Proteica en Hélice alfaRESUMEN
P-type ATPases transport ions across biological membranes against concentration gradients and are essential for all cells. They use the energy from ATP hydrolysis to propel large intramolecular movements, which drive vectorial transport of ions. Tight coordination of the motions of the pump is required to couple the two spatially distant processes of ion binding and ATP hydrolysis. Here, we review our current understanding of the structural dynamics of P-type ATPases, focusing primarily on Ca2+ pumps. We integrate different types of information that report on structural dynamics, primarily time-resolved fluorescence experiments including single-molecule Förster resonance energy transfer and molecular dynamics simulations, and interpret them in the framework provided by the numerous crystal structures of sarco/endoplasmic reticulum Ca2+-ATPase. We discuss the challenges in characterizing the dynamics of membrane pumps, and the likely impact of new technologies on the field.
Asunto(s)
Bombas Iónicas/química , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/química , Adenosina Trifosfato/metabolismo , Animales , Calcio/metabolismo , Catálisis , Humanos , Hidrólisis , Bombas Iónicas/metabolismo , Isoenzimas/química , Isoenzimas/metabolismo , Fosforilación , Unión Proteica , Conformación Proteica , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismoRESUMEN
Intrinsic disorder is common in integral membrane proteins, particularly in the intracellular domains. Despite this observation, these domains are not always recognized as being disordered. In this review, we will discuss the biological functions of intrinsically disordered regions of membrane proteins, and address why the flexibility afforded by disorder is mechanistically important. Intrinsically disordered regions are present in many common classes of membrane proteins including ion channels and transporters; G-protein coupled receptors (GPCRs), receptor tyrosine kinases and cytokine receptors. The functions of the disordered regions are many and varied. We will discuss selected examples including: (1) Organization of receptors, kinases, phosphatases and second messenger sources into signaling complexes. (2) Modulation of the membrane-embedded domain function by ball-and-chain like mechanisms. (3) Trafficking of membrane proteins. (4) Transient membrane associations. (5) Post-translational modifications most notably phosphorylation and (6) disorder-linked isoform dependent function. We finish the review by discussing the future challenges facing the membrane protein community regarding protein disorder.
Asunto(s)
Proteínas Intrínsecamente Desordenadas/metabolismo , Proteínas de la Membrana/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Humanos , Proteínas Intrínsecamente Desordenadas/química , Proteínas de la Membrana/química , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Transducción de SeñalRESUMEN
Approximately 30% of the ATP generated in the living cell is utilized by P-type ATPase primary active transporters to generate and maintain electrochemical gradients across biological membranes. P-type ATPases undergo large conformational changes during their functional cycle to couple ATP hydrolysis in the cytoplasmic domains to ion transport across the membrane. The Ca(2+)-ATPase from Listeria monocytogenes, LMCA1, was found to be a suitable model of P-type ATPases and was engineered to facilitate single-molecule FRET studies of transport-related structural changes. Mutational analyses of the endogenous cysteine residues in LMCA1 were performed to reduce background labeling without compromising activity. Pairs of cysteines were introduced into the optimized low-reactivity background, and labeled with maleimide derivatives of Cy3 and Cy5 resulting in site-specifically double-labeled protein with moderate activity. Ensemble and confocal single-molecule FRET studies revealed changes in FRET distribution related to structural changes during the transport cycle, consistent with those observed by X-ray crystallography for the sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA). Notably, the cytosolic headpiece of LMCA1 was found to be distinctly more compact in the E1 state than in the E2 state. Thus, the established experimental system should allow future real-time FRET studies of the structural dynamics of LMCA1 as a representative P-type ATPase.
Asunto(s)
ATPasas Transportadoras de Calcio/genética , ATPasas Transportadoras de Calcio/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Listeria monocytogenes/enzimología , Ingeniería de Proteínas , ATPasas Transportadoras de Calcio/química , Maleimidas/química , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Conformación ProteicaRESUMEN
Many intrinsically disordered proteins fold upon binding to other macromolecules. The secondary structure present in the well-ordered complex is often formed transiently in the unbound state. The consequence of such transient structure for the binding process is, however, not clear. The activation domain of the activator for thyroid hormone and retinoid receptors (ACTR) is intrinsically disordered and folds upon binding to the nuclear coactivator binding domain (NCBD) of the CREB binding protein. A number of mutants was designed that selectively perturbs the amount of secondary structure in unbound ACTR without interfering with the intermolecular interactions between ACTR and NCBD. Using NMR spectroscopy and fluorescence-monitored stopped-flow kinetic measurements we show that the secondary structure content in helixâ 1 of ACTR indeed influences the binding kinetics. The results thus support the notion of preformed secondary structure as an important determinant for molecular recognition in intrinsically disordered proteins.
Asunto(s)
Proteínas Intrínsecamente Desordenadas/metabolismo , Ligandos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/química , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Humanos , Proteínas Intrínsecamente Desordenadas/química , Cinética , Mutación , Resonancia Magnética Nuclear Biomolecular , Coactivador 3 de Receptor Nuclear/química , Coactivador 3 de Receptor Nuclear/genética , Coactivador 3 de Receptor Nuclear/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Biomolecular condensates are increasingly recognized as important drivers of cellular function; their dysregulation leads to pathology and disease. We discuss three questions in terms of the impending utility of data-driven techniques to predict condensate-driven biological outcomes, i.e., the impact of cellular state changes on condensates, the effect of condensates on biochemical processes within, and condensate properties that result in cellular dysregulation and disease.
RESUMEN
Many proteins do not fold into a fixed three-dimensional structure, but rather function in a highly disordered state. These intrinsically disordered proteins pose a unique challenge to protein engineering and design: How can proteins be designed de novo if not by tailoring their structure? Here, we will review the nascent field of design of intrinsically disordered proteins with focus on applications in biotechnology and medicine. The design goals should not necessarily be the same as for de novo design of folded proteins as disordered proteins have unique functional strengths and limitations. We focus on functions where intrinsically disordered proteins are uniquely suited including disordered linkers, desiccation chaperones, sensors of the chemical environment, delivery of pharmaceuticals, and constituents of biomolecular condensates. Design of functional intrinsically disordered proteins relies on a combination of computational tools and heuristics gleaned from sequence-function studies. There are few cases where intrinsically disordered proteins have made it into industrial applications. However, we argue that disordered proteins can perform many roles currently performed by organic polymers, and that these proteins might be more designable due to their modularity.
Asunto(s)
Proteínas Intrínsecamente Desordenadas , Ingeniería de ProteínasRESUMEN
α-Synuclein (α-syn) accumulates as insoluble amyloid but also forms soluble α-syn oligomers (αSOs), thought to be even more cytotoxic than fibrils. To detect and block the unwanted activities of these αSOs, we have raised 30 monoclonal antibodies (mAbs) against different forms of αSOs, ranging from unmodified αSOs to species stabilized by lipid peroxidation products and polyphenols, αSOs formed by C-terminally truncated α-syn, and multivalent display of α-syn on capsid virus-like particles (cVLPs). While the mAbs generally show a preference for αSOs, they also bind fibrils, but to variable extents. Overall, we observe great diversity in the mAbs' relative affinities for monomers and αSOs, varied requirements for the C-terminal extension of α-syn, and only a modest effect on α-syn fibrillation. Several mAbs show several orders of magnitude preference for αSOs over monomers in in-solution studies, while the commercial antibody MJF14 only bound 10-fold more strongly to αSOs than monomeric α-syn. Gratifyingly, seven mAbs almost completely block αSO permeabilization of membrane vesicles. Five selected mAbs identified α-syn-related pathologies like Lewy bodies (LBs) and Lewy Neurites, as well as Glial Cytoplasmic Inclusions in postmortem brains from people diagnosed for PD, dementia with LBs or multiple system atrophy, although to different extents. Three mAbs were particularly useful for pathological evaluation of postmortem brain human tissue, including early stages of PD. Although there was no straightforward connection between the mAbs' biophysical and immunohistochemical properties, it is encouraging that this comprehensive collection of mAbs able to recognize different aggregated α-syn species in vitro also holds diagnostic potential.
RESUMEN
Intrinsically disordered proteins are renowned for their structural plasticity when they undergo coupled folding and binding to partner proteins. The nuclear coactivator binding domain of CBP is a remarkable example of this adaptability as it folds into two different conformations depending on the binding partner. To understand the role of the conformational ensemble for plasticity in ligand recognition, we investigated the millisecond dynamics of this domain using relaxation dispersion NMR spectroscopy. All NMR signals originating from the domain are broadened, demonstrating that the whole domain experience conformational exchange. The dispersion data can be described by a global two-state exchange process between a ground state and an excited state populated to 8%. The three helices are still folded in the excited state but have a different packing from the ground state; the contact between helices 2 and 3 found in the ground state is broken in the excited state, and a new one is formed between helices 1 and 3. This suggests that while NCBD in the ground state has a structure similar to the complex with the ligand ACTR, the conformation of NCBD in the excited state has some similarity with that of NCBD in complex with the ligand IRF-3. The energy landscape of this domain is thus proposed to resemble the fold-switching proteins that have two coexisting native states, which may serve as a starting point for binding via conformational selection.
Asunto(s)
Proteína de Unión a CREB/química , Proteína de Unión a CREB/metabolismo , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Animales , Interacciones Hidrofóbicas e Hidrofílicas , Factor 3 Regulador del Interferón/química , Factor 3 Regulador del Interferón/metabolismo , Ligandos , Ratones , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Coactivador 3 de Receptor Nuclear/química , Coactivador 3 de Receptor Nuclear/metabolismo , Conformación Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Electricidad Estática , TermodinámicaRESUMEN
The urokinase-type plasminogen activator receptor (uPAR) provides a rendezvous between proteolytic degradation of the extracellular matrix and integrin-mediated adhesion to vitronectin. These processes are, however, tightly linked because the high affinity binding of urokinase regulates the binding of uPAR to matrix-embedded vitronectin. Although crystal structures exist to define the corresponding static bi- and trimolecular receptor complexes, it is evident that the dynamic property of uPAR plays a decisive role in its function. In the present study, we combine small angle x-ray scattering, hydrogen-deuterium exchange, and surface plasmon resonance to develop a structural model describing the allosteric regulation of uPAR. We show that the flexibility of its N-terminal domain provides the key for understanding this allosteric mechanism. Importantly, our model has direct implications for understanding uPAR-assisted cell adhesion and migration as well as for translational research, including targeted intervention therapy and non-invasive tumor imaging in vivo.
Asunto(s)
Matriz Extracelular , Proteolisis , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Vitronectina , Regulación Alostérica , Animales , Adhesión Celular , Línea Celular , Movimiento Celular , Medición de Intercambio de Deuterio , Drosophila melanogaster , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Humanos , Invasividad Neoplásica , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/patología , Unión Proteica , Estructura Terciaria de Proteína , Receptores del Activador de Plasminógeno Tipo Uroquinasa/química , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Dispersión de Radiación , Relación Estructura-Actividad , Vitronectina/química , Vitronectina/metabolismo , Rayos XRESUMEN
Intrinsically disordered proteins (IDPs) are widespread and important in biology but defy the classical protein structure-function paradigm by being functional in the absence of a stable, folded conformation. Here we investigate the coupling between transient secondary and tertiary structure in the protein activator for thyroid hormone and retinoid receptors (ACTR) by rationally modulating the helical propensity of a partially formed α-helix via mutations. Eight mutations predicted to affect the population of a transient helix were produced and investigated by NMR spectroscopy. Chemical shift changes distant to the mutation site are observed in regions containing other transient helices indicating that distant helices are stabilized through long-range hydrophobic helix-helix interactions and demonstrating the coupling of transient secondary and tertiary structure. The long-range structure of ACTR is also probed using paramagnetic relaxation enhancements (PRE) and residual dipolar couplings, which reveal an additional long-range contact between the N- and C-terminal segments. Compared to residual dipolar couplings and PRE, modulation of the helical propensity by mutagenesis thus reveals a different set of long-range interactions that may be obscured by stronger interactions that dominate other NMR measurements. This approach thus offers a complementary and generally applicable strategy for probing long-range structure in disordered proteins.
Asunto(s)
Proteínas Intrínsecamente Desordenadas/química , Modelos Moleculares , Conformación ProteicaRESUMEN
Single-molecule confocal microscopy experiments require concentrations which are low enough to guarantee that, on average, less than one single molecule resides in the probe volume at any given time. Such concentrations are, however, significantly lower than the dissociation constants of many biological complexes which can therefore dissociate under single-molecule conditions. To address the challenge of observing weakly bound complexes in single-molecule experiments in solution, we have designed a microfluidic device that rapidly dilutes samples by up to one hundred thousand times, allowing the observation of unstable complexes before they dissociate. The device can interface with standard biochemistry laboratory experiments and generates a spatially uniform dilution that is stable over time allowing the quantification of the relative concentrations of different molecular species.