Probing Single Biomolecules in Solution Using the Anti-Brownian Electrokinetic (ABEL) Trap

Abstract

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.

Direct single-molecule measurements of phycocyanobilin photophysics in monomeric C-phycocyanin

“…The ABEL trap suppresses the Brownian motion of single particles in free solution using electrophoretic or electroosmotic drift forces to continuously restore the particle toward the trap center (41) (Fig. 1C and SI Appendix, Fig.…”

Dissecting pigment architecture of individual photosynthetic antenna complexes in solution

“…To interrogate the photophysical states of APC in a nonperturbative solution environment, we use the ABEL trap (18,19), which integrates high-speed fluorescence tracking, feedback control, and microfluidics to counteract Brownian motion in aqueous solution. The ABEL trap enables >1,000 times longer observation time (approximately seconds) of single biomolecules in solution (20) …”

Single-molecule spectroscopy reveals photosynthetic LH2 complexes switch between emissive states

“…Indeed, modeling of these two sets of previous room temperature experiments yielded conflicting results, which the authors suggest may arise from differing immobilization schemes (14). Here, we remove this uncertainty by studying single LH2 complexes in solution to access the native heterogeneity and photodynamics (6,15).…”

“…Therefore, only through single-molecule spectroscopy can we investigate the photodynamics of individual PPCs, specifically how the absorption, energy transport, and dissipation of each change with time. We apply a room-temperature, singlemolecule technique, the anti-Brownian electrokinetic (ABEL) trap (5,6), to reveal static and dynamic heterogeneity for the primary antenna PPC of purple bacteria, light-harvesting complex 2 (LH2), without surface attachment or encapsulation.…”