Probing single biomolecules in solution using the anti-Brownian electrokinetic (ABEL) trap

  • Posted on: 10 July 2014
  • By: admin
TitleProbing single biomolecules in solution using the anti-Brownian electrokinetic (ABEL) trap
Publication TypeJournal Article
Year of Publication2012
AuthorsWang Q, Goldsmith R.H, Jiang Y, Bockenhauer SD, Moerner W.E
JournalAcc. Chem. Res.
Volume45
Pagination1955-1964
ISBN Number0001-4842
Accession Number2012:737125
KeywordsBiochemical compounds Role: ANT (Analyte), BSU (Biological study, unclassified), PEP (Physical, engineering or chemical process), PRP (Properties), ANST (Analytical study), BIOL (Biological study), PROC (Process) (probing single biomols. in soln. using, Brownian motion (translational, Diffusion (Brownian, Electroosmosis, Flow analysis (microfluidic, Fluorescence microscopy, Lab-on-a-chip, Macromolecular compounds Role: ANT (Analyte), BSU (Biological study, unclassified), PEP (Physical, engineering or chemical process), PRP (Properties), ANST (Analytical study), BIOL (Biological study), PROC (Process) (biol., Microfluidic devices, probing single biomols. in soln. using anti-Brownian electrokinetic (ABEL) trap), review single biomol anti Brownian electrokinetic trap fluorescence microscopy, Single molecule detection (probing single biomols. in soln. using anti-Brownian electrokinetic (ABEL) trap), Trapping apparatus (ABEL (anti-Brownian electrokinetic) trap
Abstract

A review. Single-mol. fluorescence measurements allow researchers to study asynchronous dynamics and expose mol.-to-mol. structural and behavioral diversity, which contributes to the understanding of biol. macromols. To provide measurements that are most consistent with the native environment of biomols., researchers would like to conduct these measurements in the soln. phase if possible. However, diffusion typically limits the observation time to ∼1 ms in many soln.-phase single-mol. assays. Although surface immobilization is widely used to address this problem, this process can perturb the system being studied and contribute to the obsd. heterogeneity. Combining the tech. capabilities of high-sensitivity single-mol. fluorescence microscopy, real-time feedback control and electrokinetic flow in a microfluidic chamber, the authors have developed a device called the anti-Brownian electrokinetic (ABEL) trap to significantly prolong the observation time of single biomols. in soln. The authors have applied the ABEL trap method to explore the photodynamics and enzymic properties of a variety of biomols. in aq. soln. 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 the authors can ext. in studies of single biomols. 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 mol. discontinuously converts from one emission/lifetime level to another in a primarily correlated way, it undergoes a series of state changes. The authors studied the ATP binding stoichiometry of the multi-subunit chaperonin enzyme TRiC in the ABEL trap by counting the no. of hydrolyzed Cy3-ATP using stepwise photobleaching. Unlike ensemble measurements, the obsd. ATP no. distributions depart from the std. 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 mols. 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 redn. of a nearby type I Cu center, the authors probed the enzymic cycle of the redox enzyme nitrate reductase. The authors detd. the rate consts. of a model of the underlying kinetics through an anal. of the dwell times of the high/low intensity levels of the fluorophore vs. nitrite concn. [on SciFinder(R)]

Alternate JournalAccounts of Chemical Research