Single Molecule Projects
Researchers: Aaron F. Phillips, Manuel A. Palacios, Elena Benito-Peña, Marcin J. Rojek
Past member: Dr. David M. Rissin (now at Quanterix), Dr. Hans-Heiner Gorris (now at University of Regensburg, Germany), Dr. Zhaohui Li (now at the Pacific Northwest National Laboratories)
Single enzyme molecule detection
Single molecule measurements provide unique information about heterogeneous molecular behaviors that are hidden using bulk methods. We separated and enclosed single β-galactosidase molecules with the fluorogenic substrate resorufin- β-galactopyranoside in an array of 50,000 femtoliter-sized chambers on the distal end of an etched optical fiber bundle. According to Poisson statistics an appropriate low enzyme concentration (1 enzyme molecule in 20 microchambers) ensures that the microchambers contain a maximum of only a single enzyme molecule while the rest are empty (Nano Letters, 2006). Trapping single enzyme molecules resolves any problems inherent to surface attachment of the enzyme. The catalytic activity of each enzyme molecule, which resulted in the production of fluorescent resorufin, was detected individually by epifluorescence microscopy (Figure 1). This new single molecule detection method is now pursued commercially by Quanterix, a company that has licensed the technology from Tufts.
Figure 1: Illustration of single enzyme molecule substrate turnover in a fiber bundle array. A schematic section of a 2 mm diameter glass optical fiber bundle containing 50,000 fibers in total is shown in the upper panel. 46-femtoliter microchambers were etched homogenously into the distal end (d) of the fibers. The fiber core is shown in white and the cladding in gray. The fiber bundle was mounted on a custom-built upright epifluorescence microscope and the reaction progress was monitored through the proximal side (p) of the fiber bundle after the microchambers had been sealed by a silicone gasket (gasket not shown). The single enzyme molecules in the microchambers convert a non-fluorescent substrate to fluorescent resorufin (yellow chambers) (© PNAS, 2007).
Activity distribution of single enzyme molecules
With our novel detection platform for single enzyme molecules we showed that individual β-galactosidase molecules exhibit a distinct catalytic activity which is broadly distributed (“static heterogeneity”) (JACS, 2008). In each experiment hundreds of individual enzyme molecules in the fiber bundle array were observed simultaneously – enough data for a thorough statistical analysis of single enzyme molecule kinetics (Figure 2).
Figure 2: Single molecule substrate turnover distribution histograms at substrate concentrations of 25 μM (blue), 50 μM (red), and 150 μM (green). Left: normalized frequency of substrate turnovers (s-1); right: normalized frequency of normalized substrate turnovers (© JACS, 2008).
The frequency distributions of the normalized velocity of an individual enzyme molecule (vi/<v>) superimpose at substrate concentrations of 25, 50, and 150 µM and have the same coefficient of variation (30 ± 1 %) (Figure 2, right). Therefore, the velocity distribution can be assumed to be a universal function of [S], and - as vi is directly proportional to kcat but not to KM - the single-molecule Michaelis-Menten equation
indicates that variability in kcat but not KM
is the source for the wide distribution of vi.
Single enzyme molecule inhibition
When we added the slow-binding inhibitor D-galactal to the enzyme/substrate solution in the microchamber array (Figure 1), we were able to observe for the first enzyme inhibition kinetics at the single molecule level (PNAS, 2007). Inhibited and active states of β-galactosidase could be clearly distinguished and the large array size provided very good statistics. With a pre-steady-state experiment, we demonstrated the stochastic character of inhibitor release, which obeys first-order kinetics (Figure 3).
Figure 3: Inhibitor release from single enzyme molecules in a pre-steady-state experiment. β-galactosidase was first saturated with the inhibitor D-galactal. The inhibitor was then 1000-fold diluted such that the inhibitor concentration was too low to rebind to the catalytic sites of the enzyme in a significant amount. The diluted solution was quickly enclosed in the array and sequential fluorescence images of single enzyme substrate turnovers were taken every 30 s. (A) A sequence of these images recorded after closing the chambers (left panel), 1020 s (middle panel), and 1920 s (right panel) shows a delayed onset of substrate turnover that we attribute to stochastic inhibitor release events. (B) Trajectories of fluorescence increase in the indicated microchambers. An empty chamber shows a constant background (red curve). (C) Distribution of off-times with 2 min binning time. (D) The semi-logarithmic plot illustrates a first-order release of inhibitor (© PNAS, 2007).
Under steady-state conditions, the quantitative detection of substrate turnover changes over long time periods revealed repeated inhibitor binding and release events, which are accompanied by conformational changes of the enzyme’s catalytic site. We proved that the rate constants of inhibitor release and binding derived from stochastic changes in the substrate turnover are consistent with bulk reaction kinetics.
Single molecule kinetics of HRP
In contrast to β-galactosidase, horseradish peroxidase (HRP) exhibits product formation rates that are on average 10 times lower at the single molecule level in the femtoliter array than in bulk solution. The redox-reaction mechanism of HRP involves two separate steps of product formation (Figure 4). HRP first oxidizes Amplex Red to non-fluorescent radical intermediates, which subsequently undergo an enzyme independent dismutation reaction to form fluorescent resorufin. If HRP is confined in a femtoliter chamber the dismutation reaction decreases such that less product is formed. This two-step oxidation mechanism of the widely used Amplex Red and other fluorogenic substrates is often overlooked. It is important for single-molecule studies with HRP as well as bulk reactions at low substrate turnover rates.
Figure 4: HRP (purple) catalyzes a one-electron oxidation (red) of non-fluorescent Amplex Red to the non-fluorescent Amplex Red radical. The formation of fluorescent resorufin (yellow) from two Amplex Red radicals is an enzyme independent dismutation reaction. The overall reaction stoichiometry between Amplex Red and H2O2 (green) is 1 : 1 only if all Amplex Red radicals are converted to resorufin (© JACS, 2009).
Single DNA molecule detection
In an alternative approach, the array surface was modified with biotin which can efficiently capture single molecules of streptavidin-labeled β-galactosidase (JACS, 2006). Experiments are under way to use the streptavidin-labeled β-galactosidase as a reporter for the detection of single DNA molecules (Figure 5). After the feasibility of this novel detection method for single DNA molecules has been clearly shown, a range of potential applications opens up for the future.
Figure 5: Illustration of single DNA molecule detection in the fiber array. Single-stranded DNA molecules were first immobilized on the microchamber surface of an etched glass fiber bundle. Upon hybridization with a biotin-labeled complementary DNA-strand and ensuing binding of streptavidin-labeled β-galactosidase, single molecules of DNA were detectable by the enzyme’s substrate turnover.
Publications
“Mechanistic aspects of horseradish peroxidase elucidated through single-molecule studies”,
H.H. Gorris, D.R. Walt, JACS 2009 in press
“Detection of single-molecule DNA hybridization using enzymatic amplification in an array of femtoliter-sized reaction vessels”,
Z. Li, R.B. Hayman, D.R. Walt, JACS 2008 130 (38), 12622-12623
"Distinct and Long-lived Activity States of Single Enzyme Molecules",
D.M. Rissin, H.H. Gorris, D.R. Walt, JACS, 2008 130 (15), 5349-5353
News highlights about this article:
a) "Distinctive Individualism", Science, Editors' Choice April 2008,
320 (5872), p. 23
. ........b) Faculty of 1000 Biology by Stuart S. Licht (7 Apr 2008)
..........http://www.f1000biology.com/article/id/1104680/evaluation
“Stochastic inhibitor release and binding from single-enzyme molecules”,
H.H. Gorris, D.M. Rissin, D.R. Walt, PNAS, 2007, 104 (45), 17680-17685
News highlights about this article:
a) “Observing single enzymes at work”, Chem. Eng. News, 2007, 85 (44), 8
b) “High-throughput, single-enzyme assay system”, Anal. Chem., 2008, 80 (1), 5
“Digital concentration readout of single enzyme molecules using femtoliter arrays and Poisson statistics”,
D.M. Rissin, D.R. Walt. Nano Letters 2006 6 (3), 520-523
“Digital readout of target binding with attomole detection limits via enzyme amplification in femtoliter arrays”,
D.M. Rissin, D.R. Walt, JACS, 2006 128 (19), 6286-6287
............News highlight about this article:
...........“Single-molecule binding assay in femtoliter arrays”, Anal. Chem., 2006, 78 (13), 4241
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