Single Enzyme Molecule Research Articles

Elastic deformations of the rotary double motor of single FoF1-ATP synthases detected in real time by Förster resonance energy transfer

Elastic conformational changes of the protein backbone are essential for catalytic activities of enzymes. To follow relative movements within the protein, Förster-type resonance energy transfer (FRET) between two specifically attached fluorophores can be applied. FRET provides a precise ruler between 3 and 8nm with subnanometer resolution. Corresponding submillisecond time resolution is sufficient to identify conformational changes in FRET time trajectories. Analyzing single enzymes circumvents the need for synchronization of various conformations. FOF1-ATP synthase is a rotary double motor which catalyzes the synthesis of adenosine triphosphate (ATP). A proton-driven 10-stepped rotary FO motor in the Escherichia coli enzyme is connected to a 3-stepped F1 motor, where ATP is synthesized. To operate the double motor with a mismatch of step sizes smoothly, elastic deformations within the rotor parts have been proposed by W. Junge and coworkers. Here we extend a single-molecule FRET approach to observe both rotary motors simultaneously in individual FOF1-ATP synthases at work. We labeled this enzyme with two fluorophores specifically, that is, on the ε- and c-subunits of the two rotors. Alternating laser excitation was used to select the FRET-labeled enzymes. FRET changes indicated associated transient twisting within the rotors of single enzyme molecules during ATP hydrolysis and ATP synthesis. Supported by Monte Carlo simulations of the FRET experiments, these studies reveal that the rotor twisting is greater than 36° and is largely suppressed in the presence of the rotation inhibitor DCCD. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).

Arrhenius Plot for a Reaction Catalyzed by a Single Molecule of β-Galactosidase

The activity of a single enzyme molecule of Escherichia coli β-galactosidase was measured using a capillary electrophoresis continuous flow assay. As the enzyme molecule traversed the capillary the incubation temperature was increased from 27 to 37 °C, providing a continuous record of the change in rate with temperature. This data was used to develop a single enzyme molecule Arrhenius plot, from which the activation energy of the reaction was determined to be 31 kJ mol(-1).

Single-Molecule Enzyme Dynamics of Monomeric Sarcosine Oxidase in a Gold-Based Zero-Mode Waveguide

The localization of optical fields is a powerful method of reducing spectroscopic background signals, enabling studies of single fluorescent molecules. Zero-mode waveguides (ZMWs) strongly confine optical fields to zeptoliter (zL, 10(-21) L) volumes and can be coupled with fluorescence microscopy to study the dynamics of single enzyme molecules due to their excellent optical confinement, precise positioning, and massive parallelism. The experiments described here exploit arrays of gold-based (Au-based) nanopores derivatized with single copies of the redox enzyme monomeric sarcosine oxidase (MSOX). MSOX contains a covalently bound flavin adenine dinucleotide (FAD) cofactor, which is highly fluorescent in the oxidized state and dark in the reduced state, thus producing a characteristic on-off fluorescence signal synchronous with transitions between oxidation states. Although aluminum (Al) is the common choice for the metallic overlayer in ZMW construction, Au is used here to access its unique surface-binding chemistry. In particular, the signal-to-noise ratio is improved for Au-based ZMWs by selective Au passivation. For MSOX reactions involving both the nominal substrate (sarcosine) and an analogous substrate (proline), statistical analysis of single-molecule temporal trajectories reveals the static heterogeneity of single-enzyme reaction rates, but no dynamic disorder. In addition, the single-molecule data confirm the independence of reduction and oxidation reactions. These structures open the way for systematic studies of the effect of molecular crowding on enzyme dynamics.

Manipulating Protein Conformations by Single-Molecule AFM-FRET Nanoscopy

Combining atomic force microscopy and fluorescence resonance energy transfer spectroscopy (AFM-FRET), we have developed a single-molecule AFM-FRET nanoscopy approach capable of effectively pinpointing and mechanically manipulating a targeted dye-labeled single protein in a large sampling area and simultaneously monitoring the conformational changes of the targeted protein by recording single-molecule FRET time trajectories. We have further demonstrated an application of using this nanoscopy on manipulation of single-molecule protein conformation and simultaneous single-molecule FRET measurement of a Cy3-Cy5-labeled kinase enzyme, HPPK (6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase). By analyzing time-resolved FRET trajectories and correlated AFM force pulling curves of the targeted single-molecule enzyme, we are able to observe the protein conformational changes of a specific coordination by AFM mechanic force pulling.

Probing single-molecule enzyme active-site conformational state intermittent coherence.

The relationship between protein conformational dynamics and enzymatic reactions has been a fundamental focus in modern enzymology. Using single-molecule fluorescence resonance energy transfer (FRET) with a combined statistical data analysis approach, we have identified the intermittently appearing coherence of the enzymatic conformational state from the recorded single-molecule intensity-time trajectories of enzyme 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) in catalytic reaction. The coherent conformational state dynamics suggests that the enzymatic catalysis involves a multistep conformational motion along the coordinates of substrate-enzyme complex formation and product releasing, presenting as an extreme dynamic behavior intrinsically related to the time bunching effect that we have reported previously. The coherence frequency, identified by statistical results of the correlation function analysis from single-molecule FRET trajectories, increases with the increasing substrate concentrations. The intermittent coherence in conformational state changes at the enzymatic reaction active site is likely to be common and exist in other conformation regulated enzymatic reactions. Our results of HPPK interaction with substrate support a multiple-conformational state model, being consistent with a complementary conformation selection and induced-fit enzymatic loop-gated conformational change mechanism in substrate-enzyme active complex formation.

Single Molecule Assay of Escherichia coli β-Galactosidase Using Two Competing Substrates Simultaneously, DDAO-β-D-Galactoside and Resorufin-β-D-Galactoside

Single enzyme molecule assays were performed on E. coli β-galactosidase using a capillary electrophoresis-based protocol. Assays were performed using double incubations and two substrates, resorufin-β-D-galactoside and DDAO-β-D-galactoside, simultaneously. The variation between individual enzyme molecules in the ratio of product peak areas for the two different substrates used was indistinguishable from the variation in peak areas of the replicate incubations for a given enzyme molecule. This suggests that the enzyme is not heterogeneous with respect to its relative activity with the two different substrates used.

Correction to “Dynamic Simulations of Single Molecule Enzyme Networks”

Correction to “Dynamic Simulations of Single Molecule Enzyme Networks”

Cellobiohydrolase Hydrolyzes Crystalline Cellulose on Hydrophobic Faces

Biodegradation of plant biomass is a slow process in nature, and hydrolysis of cellulose is also widely considered to be a rate-limiting step in the proposed industrial process of converting lignocellulosic materials to biofuels. It is generally known that a team of enzymes including endo- and exocellulases as well as cellobiases are required to act synergistically to hydrolyze cellulose to glucose. The detailed molecular mechanisms of these enzymes have yet to be convincingly elucidated. In this report, atomic force microscopy (AFM) is used to image in real-time the structural changes in Valonia cellulose crystals acted upon by the exocellulase cellobiohydrolase I (CBH I) from Trichoderma reesei. Under AFM, single enzyme molecules could be observed binding only to one face of the cellulose crystal, apparently the hydrophobic face. The surface roughness of cellulose began increasing after adding CBH I, and the overall size of cellulose crystals decreased during an 11-h period. Interestingly, this size reduction apparently occurred only in the width of the crystal, whereas the height remained relatively constant. In addition, the measured cross-section shape of cellulose crystal changed from asymmetric to nearly symmetric. These observed changes brought about by CBH I action may constitute the first direct visualization supporting the idea that the exocellulase selectively hydrolyzes the hydrophobic faces of cellulose. The limited accessibility of the hydrophobic faces in native cellulose may contribute significantly to the rate-limiting slowness of cellulose hydrolysis.

Single-Molecule Enzymology: Studies on Glutathione-S-Transferases

An emerging concept in enzymology is the catalytic transition state ensemble: instead of the single, well-defined transition state (TS) of classical theory, enzymatic reactions progress through multiple ‘valleys’ in the energy landscape, each with its own TS. Long-lived conformations of the enzyme-substrate (ES) complex would take different pathways through the TS ensemble, and would therefore result in functional heterogeneity (i.e., a distribution of catalytic rate constants). The functional properties of enzymes are therefore completely controlled by the nature of the energetic landscape for the ES complex and the TS ensemble. We have developed TIRFM (total internal reflection fluorescence microscopy) and FCS (fluorescence correlation spectroscopy) methods to measure turnover by single immobilized enzyme molecules. We applied these methods, complemented by molecular dynamics simulations and ab initio calculations, to two isoforms of glutathione-S-transferase (GST) to examine the validity of the TS ensemble model. GST isoform A1-1 is an abundant hepatic enzyme involved in detoxification, and is extremely promiscuous in its substrate range. GST A4-4 is the predominant isoform in the heart and brain, and is specific towards lipid peroxidation products. Contrasting the behavior of these two enzymes will address fundamental questions about the mechanisms of substrate promiscuity.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Activator-induced dynamic disorder and molecular memory in human two-pore domain hTREK1 K+ channel

Ion channels are fundamental molecules in the nervous system that catalyze the flux of ions across the cell membrane. Ion channel flux activity is comparable to the catalytic activity of enzyme molecules. Saturating concentrations of substrate induce "dynamic disorder" in the kinetic rate processes of single-enzyme molecules and consequently, develop correlative "memory" of the previous history of activities. Similarly, binding of ions as substrate alone or in presence of agonists affects the catalytic turnover of single-ion channels. Here, we investigated the possible existence of dynamic disorder and molecular memory in the single human-TREK1-channel due to binding of substrate/agonist using the excised inside-out patch-clamp technique. Our results suggest that the single-hTREK1-channel behaves as a typical Michaelis-Menten enzyme molecule with a high-affinity binding site for K(+) ion as substrate. But, in contrast to enzyme, dynamic disorder in single-hTREK1-channel was not induced by substrate K(+) binding, but required allosteric modification of the channel molecule by the agonist, trichloroethanol. In addition, interaction of trichloroethanol with hTREK1 induced strong correlation in the waiting time and flux intensity, exemplified by distinct mode-switching between high and low flux activities. This suggested the induction of molecular memory in the channel molecule by the agonist, which persisted for several decades in time. Our mathematical modeling studies identified the kinetic rate processes associated with dynamic disorder. It further revealed the presence of multiple populations of distinct conformations that contributed to the "heterogeneity" and consequently, to the molecular memory phenomenon that we observed. The online version of this article (doi:10.1007/s12154-010-0053-3) contains supplementary material, which is available to authorized users.

Quantitative Analysis of Integrated Energy Metabolism of Muscle Cells: Experimental and Theoretical Studies

Quantitative analysis, in combination with detailed experimental studies, is the basis of Molecular System Bioenergetics (MSB) which takes into account specific interactions between cellular components, formation of metabolic dissipative structures and resulting system level properties such as metabolic compartmentation. There are several experimental and theoretical approaches used: studies in vivo kinetics of mitochondrial respiration regulation; Metabolic Control Analysis of integrated energy metabolism; mathematical modeling. Cardiac cells are characterized by high level of structural organization with very regular arrangement of mitochondria and their interaction with cytoskeleton. Heterodimeric tubulin containing beta II isoform is connected to mitochondrial outer membrane, specifically controls VDAC permeability and is coexpressed with mitochondrial creatine kinase. These proteins form with ATP Synthasome a supercomplex Mitochondrial Interactosome (MI) in which the kinetics of regulation of respiration by MtCK is significantly altered in comparison with mitochondria in vitro affinity for extramitochondrial ATP is strongly decreased due to limited permeability of VDAC but for creatine increased. Metabolic Control Analysis of reactions in MI showed that flux control coefficients are significantly increased for reactions of ADP/ATP recycling coupled with phosphocreatine production by MtCK. These experimental data are the basis of the mathematical model of compartmentalized energy transfer and show that about 90 % of energy is carried out of mitochondria by phosphocreatine due to specific structure of MI. This model explains well the metabolic aspects of Frank -Starling law of the heart. In cancerous HL −1 cells of cardiac phenotype MtCK and beta II tubulin are not expressed. Mathematical models based on the theory of homogenous intracellular medium and CK equilibrium do not explain adequately the experimental observations. In future development, application of single-molecule enzyme kinetics and theory of nonlinear biochemical reaction networks is necessary.

Revealing time bunching effect in single-molecule enzyme conformational dynamics

In this perspective, we focus our discussion on how the single-molecule spectroscopy and statistical analysis are able to reveal enzyme hidden properties, taking the study of T4 lysozyme as an example. Protein conformational fluctuations and dynamics play a crucial role in biomolecular functions, such as in enzymatic reactions. Single-molecule spectroscopy is a powerful approach to analyze protein conformational dynamics under physiological conditions, providing dynamic perspectives on a molecular-level understanding of protein structure-function mechanisms. Using single-molecule fluorescence spectroscopy, we have probed T4 lysozyme conformational motions under the hydrolysis reaction of a polysaccharide of E. coli B cell walls by monitoring the fluorescence resonant energy transfer (FRET) between a donor-acceptor probe pair tethered to T4 lysozyme domains involving open-close hinge-bending motions. Based on the single-molecule spectroscopic results, molecular dynamics simulation, a random walk model analysis, and a novel 2D statistical correlation analysis, we have revealed a time bunching effect in protein conformational motion dynamics that is critical to enzymatic functions. Bunching effect implies that conformational motion times tend to bunch in a finite and narrow time window. We show that convoluted multiple Poisson rate processes give rise to the bunching effect in the enzymatic reaction dynamics. Evidently, the bunching effect is likely common in protein conformational dynamics involving in conformation-gated protein functions. In this perspective, we will also discuss a new approach of 2D regional correlation analysis capable of analyzing fluctuation dynamics of complex multiple correlated and anti-correlated fluctuations under a non-correlated noise background. Using this new method, we are able to map out any defined segments along the fluctuation trajectories and determine whether they are correlated, anti-correlated, or non-correlated; after which, a cross correlation analysis can be applied for each specific segment to obtain a detailed fluctuation dynamics analysis.

The Chemical Master Equation Approach to Nonequilibrium Steady-State of Open Biochemical Systems: Linear Single-Molecule Enzyme Kinetics and Nonlinear Biochemical Reaction Networks

We develop the stochastic, chemical master equation as a unifying approach to the dynamics of biochemical reaction systems in a mesoscopic volume under a living environment. A living environment provides a continuous chemical energy input that sustains the reaction system in a nonequilibrium steady state with concentration fluctuations. We discuss the linear, unimolecular single-molecule enzyme kinetics, phosphorylation-dephosphorylation cycle (PdPC) with bistability, and network exhibiting oscillations. Emphasis is paid to the comparison between the stochastic dynamics and the prediction based on the traditional approach based on the Law of Mass Action. We introduce the difference between nonlinear bistability and stochastic bistability, the latter has no deterministic counterpart. For systems with nonlinear bistability, there are three different time scales: (a) individual biochemical reactions, (b) nonlinear network dynamics approaching to attractors, and (c) cellular evolution. For mesoscopic systems with size of a living cell, dynamics in (a) and (c) are stochastic while that with (b) is dominantly deterministic. Both (b) and (c) are emergent properties of a dynamic biochemical network; We suggest that the (c) is most relevant to major cellular biochemical processes such as epi-genetic regulation, apoptosis, and cancer immunoediting. The cellular evolution proceeds with transitions among the attractors of (b) in a “punctuated equilibrium” manner.

ChemInform Abstract: Analytical Chemistry on the Femtoliter Scale

AbstractReview: 126 refs.

Effect of Size, Quaternary Structure and Translational Error on the Static and Dynamic Heterogeneity of β-Galactosidase and Measurement of Electrophoretic Dynamic Heterogeneity

Single enzyme molecule assays were performed using capillary electrophoresis-based protocols on beta-galactosidase from Lactobacillus delbrueckii, Lactobacillus reuteri, Lactobacillus helveticus and Bacillus circulans. The enzyme was found to show static heterogeneity with respect to catalytic rate and the variance in rate increased with protein size. This is consistent with the proposal that random errors in translation may be an important underlying component of enzyme heterogeneity. Additionally these enzymes were found to show static heterogeneity with respect to electrophoretic mobility. Comparison of wild-type and rpsL E. coli beta-galactosidase expressed in the presence and absence of streptomycin suggested that increases in error do not result in detectable increases in the dynamic heterogeneity of activity with increasing temperature. Finally, a method was developed to measure the dynamic heterogeneity in electrophoretic mobility.

Substrate catalysis enhances single-enzyme diffusion.

We show that diffusion of single urease enzyme molecules increases in the presence of urea in a concentration-dependent manner and calculate the force responsible for this increase. Urease diffusion measured using fluorescence correlation spectroscopy increased by 16-28% over buffer controls at urea concentrations ranging from 0.001 to 1 M. This increase was significantly attenuated when urease was inhibited with pyrocatechol, demonstrating that the increase in diffusion was the result of enzyme catalysis of urea. Local molecular pH changes as measured using the pH-dependent fluorescence lifetime of SNARF-1 conjugated to urease were not sufficient to explain the increase in diffusion. Thus, a force generated by self-electrophoresis remains the most plausible explanation. This force, evaluated using Brownian dynamics simulations, was 12 pN per reaction turnover. These measurements demonstrate force generation by a single enzyme molecule and lay the foundation for a further understanding of biological force generation and the development of enzyme-driven nanomotors.

Heterogeneous Properties of Individual Molecules of β-Galactosidase from the Thermophilic Bacteria Geobacillus stearothermophilus

Single enzyme molecule assays were performed on beta-galactosidase from the thermophilic bacteria Geobacillus stearothermophilus using a capillary electrophoresis-based continuous flow assay and the substrate DDAO-beta-D: -galactoside. The enzyme was found to be heterogeneous with respect to catalytic rate, electrophoretic mobility and activation energy of catalysis. Catalytic rate was also found to vary over time for individual molecules at elevated temperature. Comparison with beta-galactosidase from the mesophilic bacteria Escherichia coli showed that the variation in activity over time was less pronounced and the average activation energy of catalysis was lower for the Geobacillus stearothermophilus enzyme. Attempts to measure the properties of individual beta-galactosidase molecules from the thermophilic bacteria Thermus thermophilus and the cold-adapted bacteria Pseudoalteromas haloplanktis using this assay were unsuccessful.

A single-molecule enzymatic assay in a directly accessible femtoliter droplet array

The enzyme assay in a femtoliter chamber array is a simple and efficient method for concentrating the reaction product; it greatly improves the detection sensitivity down to the single-molecule level. However, in previous methods, controlling the initiation and termination of the reaction in each chamber is difficult once enclosed. Furthermore, the recovery of the enzyme and product is also difficult. To overcome these drawbacks, we developed a femtoliter droplet array in which the individual droplets are fixed on the substrate and are directly accessible from outside. A hydrophilic-in-hydrophobic micropatterned surface was used for the preparation of the droplets. When the aqueous solution on the surface is exchanged with oil, the hydrophilic surface retains the aqueous solution, and more than 10(6) dome-shaped droplets that are usable for further assay can be prepared simultaneously. The curvature radius of the droplet obeys the Young-Laplace equation, and the volume can be precisely controlled by the micropipette, which applies pressure into the droplet. Changing the pressure makes the addition, collection, and exchange of the aqueous content for individual droplets possible. Using these advantages, we successfully measured the kinetic parameters of the single-molecule enzyme β-galactosidase and rotary motor protein F(1)-ATPase enclosed in a droplet.

Imaging Single Enzyme Molecules under In Situ Conditions

Imaging Single Enzyme Molecules under In Situ Conditions

Directed Metallization of Single-Enzyme Molecules With Preserved Enzymatic Activity

A new method for the fabrication of molecular, water-soluble, and biologically active enzyme-metal hybrids was designed and its feasibility demonstrated. The method is based on the display of nucleation sites directing a subsequent electroless deposition of palladium and other metals to the enzyme's surface. The process is carried out under mild physiological conditions, enabling the preservation of enzymatic activity and water solubility. The feasibility of the new method was demonstrated by using the enzyme glucose oxidase and palladium combination as the first model system. The glucose oxidase-palladium hybrid thus obtained retained their solubility and enzymatic glucose oxidation capabilities. Hybrids immobilized on platinum electrodes exhibited ldquonanowiringrdquo and effective direct electron transfer from the enzyme catalytic site to the electrode. The new enzyme-metal hybrids thus obtained may be readily incorporated into miniaturic biosensors and biochips, used as novel antibacterial agents or as markers for improved in vivo imaging. Furthermore, the methodology developed may be readily extended to a series of metal coatings on the surface of biologically active proteins.