G Protein-Coupled Receptors (GPCRs) comprise a large family of seven-helix transmembrane proteins which regulate cellular signaling by sensing light, ligands, and binding proteins. The GPCR activation process, however, is not a simple on-off switch; current models suggest a complex conformational landscape in which the active, signaling state includes multiple conformations with similar downstream activity. The present study probes the conformational dynamics of single β2-Adrenergic Receptors (β2ARs) in the solution phase by Anti-Brownian ELectrokinetic (ABEL) trapping. The ABEL trap uses fast electrokinetic feedback in a microfluidic configuration to allow direct observation of a single fluorescently-labeled β2AR for hundreds of milliseconds to seconds. By choosing a reporter dye and labeling site sensitive to ligand binding, we observe a diversity of discrete fluorescence intensity and lifetime levels in single β2ARs, indicating a varying radiative lifetime and a range of discrete conformational states with dwell times of hundreds of milliseconds. We find that binding of agonist increases the dwell times of these states, and furthermore, we observe millisecond fluctuations within states. The intensity autocorrelations of these faster fluctuations are well-described by stretched exponential functions with stretching exponent β ~ 0.5, suggesting protein dynamics over a range of timescales.
Conspectus Single-molecule fluorescence measurements allow researchers to study asynchronous dynamics and expose molecule-to-molecule structural and behavioral diversity, which contributes to the understanding of biological macromolecules. To provide measurements that are most consistent with the native environment of biomolecules, researchers would like to conduct these measurements in the solution phase if possible. However, diffusion typically limits the observation time to approximately one millisecond in many solution-phase single-molecule assays. Although surface immobilization is widely used to address this problem, this process can perturb the system being studied and contribute to the observed heterogeneity. Combining the technical capabilities of high-sensitivity single-molecule fluorescence microscopy, realtime feedback control and electrokinetic flow in a microfluidic chamber, we have developed a device called the Anti-Brownian ELectrokinetic (ABEL) trap to significantly prolong the observation time of single biomolecules in solution. We have applied the ABEL trap method to explore the photodynamics and enzymatic properties of a variety of biomolecules in aqueous solution and present four examples: the photosynthetic antenna allophycocyanin, the chaperonin enzyme TRiC, a G protein-coupled receptor protein, and the blue nitrite reductase redox enzyme. These examples illustrate the breadth and depth of information which we can extract in studies of single biomolecules with the ABEL trap. When confined in the ABEL trap, the photosynthetic antenna protein allophycocyanin exhibits rich dynamics both in its emission brightness and its excited state lifetime. As each molecule discontinuously converts from one emission/lifetime level to another in a primarily correlated way, it undergoes a series of state changes. We studied the ATP binding stoichiometry of the multi-subunit chaperonin enzyme TRiC in the ABEL trap by counting the number of hydrolyzed Cy3-ATP using stepwise photobleaching. Unlike ensemble measurements, the observed ATP number distributions depart from the standard cooperativity models. Single copies of detergent-stabilized G protein-coupled receptor proteins labeled with a reporter fluorophore also show discontinuous changes in emission brightness and lifetime, but the various states visited by the single molecules are broadly distributed. As an agonist binds, the distributions shift slightly toward a more rigid conformation of the protein. By recording the emission of a reporter fluorophore which is quenched by reduction of a nearby type I Cu center, we probed the enzymatic cycle of the redox enzyme nitrate reductase. We determined the rate constants of a model of the underlying kinetics through an analysis of the dwell times of the high/low intensity levels of the fluorophore versus nitrite concentration.
The peridinin-chlorophyll-protein (PCP) is an accessory light-harvesting complex found in red-tide dinoflagellates. PCP absorbs photons primarily in the blue-green spectral region via peridinin (Per) carotenoid pigments which then transfer excitations to chlorophyll (Chl) and ultimately downstream to photosystem II (PSII). Whereas the ultrafast dynamics of PCP are well-studied, much less is known about slower protein dynamics on time scales of milliseconds and seconds. Previous single-molecule studies of spectral emission and intensity have attached PCP to surfaces, but the native environment of PCP is in the lumen, meaning that a surface-attached environment could perturb its native conformations. To address this concern, we use the anti-Brownian electrokinetic (ABEL) trap to study single PCP monomers in solution for several seconds each. We measure, for the first time, simultaneous single-molecule intensity, lifetime, and spectral emission shifts for each trapped PCP monomer. The rate of reversible spectral redshifts depends linearly on irradiance over a factor of 30, indicating a light-induced mechanism which we attribute to a protein conformational change. Independent of these spectral shifts, our measurements of intensity and lifetime show reversible Chl quenching. In contrast to previous work, we show that this quenching cannot result from isolated photobleaching of Chl. These independent mechanisms arise from distinct conformational changes which maintain relatively stable fluorescence emission.
F1-ATPase is the soluble portion of the membrane-embedded enzyme FoF1-ATP synthase that catalyzes the production of adenosine triphosphate in eukaryotic and eubacterial cells. In reverse, the F1 part can also hydrolyze ATP quickly at three catalytic binding sites. Therefore, catalysis of ‘non-productive’ ATP hydrolysis by F1 (or FoF1) must be minimized in the cell. In bacteria, the ε subunit is thought to control and block ATP hydrolysis by mechanically inserting its C-terminus into the rotary motor region of F1. We investigate this proposed mechanism by labeling F1 specifically with two fluorophores to monitor the C-terminus of the ε subunit by Förster resonance energy transfer. Single F1 molecules are trapped in solution by an Anti-Brownian electrokinetic trap which keeps the FRET-labeled F1 in place for extended observation times of several hundreds of milliseconds, limited by photobleaching. FRET changes in single F1 and FRET histograms for different biochemical conditions are compared to evaluate the proposed regulatory mechanism.
In photosynthetic light harvesting, absorbed photoenergy transfers through networks of pigment-protein complexes to a central location, known as the reaction center, for conversion to chemical energy. This process occurs with remarkable near-unity quantum efficiency. Pigment-protein complexes exhibit emergent properties very different from those of their component molecules, resulting from interactions among the pigments and between the pigments and the surrounding protein environment. Thus, the precise molecular mechanisms of the energy transport process are complex and coupled, obscuring their molecular origin. Furthermore, because these interactions are sensitive to the molecular structure, they vary from complex to complex and vary in time for each individual complex due to fluctuations of the protein's conformation. To explore the effect of complex-to-complex variation, we study individual photosynthetic proteins, one at a time. We use a solution-phase, single-molecule technique, known as the Anti-Brownian Electrokinetic (ABEL) trap, to elucidate the conformational dynamics of single photosynthetic pigment-protein complexes without introducing additional perturbations from immobilization strategies. After reviewing the principles of the ABEL trap, we demonstrate its application to the study of several photosynthetic pigment-protein complexes. We demonstrate that the ABEL trap approach can lead to an increased understanding of photosynthetic complexes by presenting three examples: (1) analysis of photodegradation pathways, (2) characterization of complex-to-complex heterogeneity, and (3) identification of distinct functional forms.
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