Lorentz Center - Seeing enzymes in action from 1 Nov 2010 through 5 Nov 2010
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    Seeing enzymes in action
    from 1 Nov 2010 through 5 Nov 2010

 

Abstracts

 

 

Yann Astier

Nanopore and Nanochannel Synthesis for Molecular Interaction Characterisation at the Single-Molecule Level”

 

Our fundamental objective is to develop experimental techniques to observe molecular interactions at the single molecule level in real time. Due to their stochastic nature, real time single molecule monitoring techniques are well suited to reveal molecular interaction mechanisms. I will develop nanopores and nanochannels to help reveal the nature of their molecular interactions with chosen dissolved molecules. Nanopores are an emerging class of single-molecule sensors. They are unique because analytes can be probed without chemical modification and/or surface immobilisation, thus preserving structure/function and allowing very high throughput. Nanopores are circular openings with nm diameter through a membrane-like material. Trans-membrane ionic current measurements can be used to monitor the ion conductance of a single nanopore. Dissolved analytes can be detected if they enter and interact with the nanopore inner channel by altering its conductance. Depending on the pore dimensions, single molecules can be detected in this way. But these molecules shoot through the nanopore so rapidly that sequencing the DNA passing through them, for example, becomes a great challenge.

 

 

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Gerard Canters

“FRET-based optical and electrochemical studies of redox enzymes down to the single-molecule level”

 

 

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Jianshu Cao

“Non-equilibrium conformational fluctuations in Enzymes”

 

This talk focuses on theoretical analysis of time traces measured in single-molecule enzyme/protein experiments.  Validity of MM Equation:  Many enzymatic reactions in biochemistry are far more complex than the celebrated Michaelis-Menten  (MM) scheme, but the observed turnover rates often obey  the hyperbolic dependence on the substrate concentration.  To resolve the longstanding puzzle, we apply the flux balance method to predict the functional form of the substrate dependence in the mean turnover time of complex enzymatic reactions and identify detailed balance (DB) as a condition for the MM equation to describe the substrate dependence. SM Signatures of Detailed Balance Violations:  This above prediction can be verified in single-molecule event-averaged measurements using the recently-proposed signatures of DB violations: peaks in the single molecule waiting time distribution, broken time reversal symmetry, and a lack of diagonal features in two-dimensional contours. Non-equilibrium Enzyme Kinetics:  When the DB condition is violated, the MM expression will break down. We derive a generalized form of  the MM equation, which relates the functional form of the substrate dependence to non-equilibrium conformational currents. The generalized MM equation provides a unified approach to analyze non-MM behavior and predicts cooperativity, inhibition, and multi-stability.

Department of Chemistry, MIT, Cambridge, MA 02139

 

 

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Peng Chen

"Single-Molecule Catalysis: Beyond Enzymes"

 

Amid homogeneous, heterogeneous, and biological catalysis, enzymes are biology’s nanocatalysts that speed up chemical transformations. Advances in single-molecule techniques have made it possible to visualize enzyme actions in real time, one molecule at a time. These studies have generated much insight into the mechanism and dynamics of these bio-nanocatalysts. In this talk I will present our single-molecule studies of nanocatalysts in the regime of heterogeneous catalysis, namely metal nanoparticle catalysts. As compared with enzymes, for which a population of molecules can be chemically identical, metal nanoparticles are intrinsically inhomogeneous and are thus particularly suited for single-molecule studies. Using single-molecule microscopy of fluorogenic reactions, we were able to monitor catalysis on metal nanoparticles at the single-particle, single-turnover resolution in real time. We obtain insights into the intricate interplay between inhomogeneous reactivity, dynamic surface restructuring, differential selectivity in parallel reaction pathways, and variable surface sites in nanoscale catalysis. Attempts are also made to draw conceptual analogies between enzyme catalysis and heterogeneous catalysis.

Peng Chen, Department of Chemistry and Chemical Biology, Cornell University

 

 

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Joerg Enderlein

"Molecular sizing by measuring translational and rotational diffusion"

 

Several decades ago, Magde, Elson and Webb invented an ingenuous method for measuring diffusion coefficients of fluorescent molecules: Fluorescence Correlation Spectroscopy (FCS). In FCS, the fluctuations of a fluorescence signal which is generated in a femtoliter-sized volume of a sample solution is analyzed by a correlation analysis. This analysis can then be used for extracting information about molecular diffusion, intramolecular conformational dynamics, intermolecular interactions, or photophysical processes. Recently, we developed a modification of conventional FCS, which is called dual-focus FCS (2fFCS), that allows for precise and absolute sizing of molecules at pico- to nanomolar concentrations. In the presentation, the method is described in detail, and manifold applications of 2fFCS will be given. Among them are: Precise sizing proteins and peptides, monitoring conformational changes of proteins, monitoring protein-protein interactions, and measuring diffusion of transmembrane proteins in lipid bilayers. In the final part of the presentation, nanosecond-FCS will be presented as a tool for measuring rotational diffusion of molecules, which is a powerful alternative for obtaining information about molecular size and shape.

 

 

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Gianfranco Gilardi

“Getting “action” from drug metabolising enzymes: exploiting the anatomy of protein assembly for amperometry

 

The ability of getting electrochemical response from drug metabolising enzymes is an attractive target not only for fundamental studies in the redox chemistry of these enzymes, but also for the development of amperometric platform useful for the discovery of new drugs. Our laboratory has turned its attention to human liver cytochromes P450 and Flavin Monoxygenase 3 (hFMO3): these 2 classes of enzymes together they cover more than 90% of the relevant drug metabolising enzymes. They all require electrons deriving from NADPH to sustain their catalytic activity, but they present difficulties in the direct interactions with electrode surfaces due to the deeply buried catalytic sites and their association with the membrane of the endoplasmic reticulum. Learning from the modular nature of proteins, we apply protein engineering approaches to tackle these problems. In human cytochromes P450 we eliminated the N-terminal membrane-binding domain and we added a fused non-physiological reductase able to productively interact with the electron surface and to sustain the catalytic activity of the heme. This has allowed the development of an amperometric platform able to measure human polymorphic response in drug metabolism, important for determination basis of personalised pharmacological dosage, and the construction of a P450-based microfluidic device. In contrast, for the hFMO3 we improved solubility by elimination of a C-term “module”, a helix that modelling studies of hFMO3 suggested to be involved in membrane binding. Data show that deletion of the helix does not affect folding, stability and substrate turnover, and this should improve the electrochemical amenability of this enzyme. Very recently we investigated the structure-function of a particular human cytochrome P450: aromatase (rArom). The relevance of aromatase is in its ability to convert androgens into estrogens and has been focus of research in the area of sexual development and breast cancer. We obtained a soluble form of rArom able to respond on electrode surfaces. In this case the “modular” nature of protein assembly has been confirmed by the 3D X-ray structure that showed a self-assembly oligomeric organisation in arrays. University of Torino, Italy

 

 

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David Grünwald

"Spectrally resolved tracking of single molecules: mRNA export across the nuclear pore"

 

Transport through the nuclear pore complex (NPC) is the only link between nucleus and cytoplasm. Transport is selective and mediated by transport factors. Export of mRNA occurs via NPCs, large nano-machines with diameters of roughly 120 nm. Hence, export of mRNA occurs over distances smaller than the optical resolution of conventional light microscopes. While there is extensive knowledge on the physical structure and composition of the NPC transport selectivity and dynamics within the NPC have not been addressed for export of mRNAs in living cells. In this work we develop a super-registration approach using fluorescence microscopy that can overcome the current limitations of colocalization by means of measuring intermolecular distances of chromatically different fluorescent molecules with nm precision. With this method we achieve 20 ms time- and 26 nm spatial precision, rendering the capture of highly transient interactions in living cells possible. With this method we were able to spatially resolve the kineic rates of mRNA transport and present a three step model consisting of docking (80ms), transport (5-20ms) and release (80ms), totalling 180±10 ms. Importantly, the translocation through the channel was not the rate-limiting step, mRNAs can move bi-directionally in the pore complex and not all pores are equally active.

Kavli Institute of NanoScience, Department of BioNanoScience, TU Delft, Lorentzweg 1, 2628 CJ Delft, Netherlands

 

 

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Gordon Hammes

“Enzymes in action: conformation-coupled catalysis”

 

The basis for the remarkable catalytic efficiency of enzymes will be discussed using dihydrofolate reductase (DHFR) as a paradigm.  DHFR has been probed with a variety of different methods, including steady state kinetics, transient kinetics, nmr, and single-molecule methods.  Emphasis will be placed on recent results obtained with single-molecule technology.  The general mechanism that has emerged is a multiple-pathway, multiple intermediate scheme involving conformational ensembles.  This mechanism, which is applicable to enzyme catalysis in general, will be described in terms of transition state theory and free energy surfaces.   In addition, a critical discussion of the application of single-molecule fluorescence methodology to enzymes will be presented for consideration by the workshop.

Gordon G. Hammes, Department of Biochemistry, Duke University, Durham, NC 27710

 

 

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Peter Hildebrandt

“Electric field effects on the protein and electron transfer dynamics of immobilized heme proteins – vibrational spectroscopic approaches”

 

Most of the biological redox processes occur at or in membranes and thus under reaction conditions that differ substantially from those in the solution phase. Specifically, high local electric fields and restricted mobility may have a substantial impact on the reaction mechanisms and dynamics as well as on the structure of the proteins. To understand these fundamental processes on a molecular level, we have employed various vibrational spectroscopic approaches that allow probing structural changes, rotational diffusion, and charge transfer reactions of proteins immobilised on biomimetic interfaces. These interfaces are designed to mimic biological membranes using self-assembled monolayers of lipid analogues on Ag or Au electrodes which serve as an amplifier for the spectroscopic signals (surface enhanced resonance Raman and surface enhanced infrared absorption spectroscopy) and as an artificial electron donor/acceptor for the immobilised redox proteins and enzymes. Moreover, these devices offer the opportunity to control the local electric field at the protein binding site which is one of the crucial parameters determining the interfacial processes. In this contribution we illustrate the potential of this experimental approach, combined with theoretical calculations, for electron transfer proteins and redox enzymes.

Technische Universität Berlin, Institut f. Chemie, Sekr. PC14, Straße des 17. Juni 136, D-10623 Berlin, Germany

References

[1] Murgida DH, Hildebrandt P, Phys. Chem. Chem. Phys. 2005, 7, 3773-3784.

[2] Murgida DH, Hildebrandt P, Chem. Soc. Rev. 2008, 37, 937-945.

 

 

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Johan Hofkens

"Single molecule enzymatics: from model systems to cells"

 

1. Introduction

Optical spectroscopy at the ultimate limit of a single molecule has grown over the past years into a powerful technique for exploring the individual nano-scale behavior of molecules in complex local environments. Observing a single molecule removes the usual ensemble average, allowing the exploration of hidden heterogeneity in complex condensed phases as well as direct observation of dynamic changes, without synchronization. In this contribution,   single molecule spectroscopy will be applied on different enzymatic systems.

2. Single molecule enzymatics on immobilized enzymes

Our approach to realize the observation of the real-time catalysis and substrate kinetics of a single-enzyme reaction is based on a simple protocol (immobilization of the enzyme on a cover glass) and a robust enzyme (lipase CalB, chymotrypsin). In order to study the catalysis by a single enzyme, we used a fluorogenic substrate. With this approach, we were able to monitor single enzyme turnovers for extended periods of time (hours). We were able to record fluorescence intensity traces from more than 10000 turnovers and 107 detected photons for one single enzyme. By analyzing the on-off steps in the recorded fluorescence intensity traces we observed a stretched exponential waiting time pdf (probability density function) in the enzymatic catalytic activity. A possible explanation regarding the origin of non-exponential character of the pdf is that the enzymatic kinetics involves a broad conformational spectrum, of which only a finite few conformations are catalytically active. The concept is broadly applicable to a variety of enzymatic systems and also delivers information on how enzymes deactivate.

3. Single molecule enzymatics of phospholipases on bilayers

Next, our attempts to follow enzyme activity and dynamics of phospho-lipases in their natural environment, eg phospho-lipid layers, with wide field microscopy will be discussed.  By labeling both enzyme and the layer, details about the mode of action for this surface active class of enzymes can be revealed.

4. Unraveling the mode of action of ADAMTS13

ADAMtS 13 is a metaloprotease that is regulating the so called von Willebrand factor ( vWF). vWF is a blood glycoprotein involved in hemostasis. Interestingly, a conformational change of the vWF (the substrate) is required for enzyme activity. The strategy developed in the laboratory to address this system at the single molecule level will be explained.

Hofkens, Department of Chemistry, KULeuven, Celestijnenlaan 200 F 3001 Heverlee, Belgium

 

 

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Randy Howard Goldsmith

“Seeing Enzymes (and Fluorescent Proteins) in Action in Solution”

 

Observation of dynamics of single biomolecules over a prolonged time without altering the biomolecule via immobilization is achieved with a specialized microfluidic device.  This device, the Anti-Brownian ELectrokinetic (ABEL) Trap, uses real-time electrokinetic feedback to cancel Brownian motion of single objects in solution. First, we use the ABEL Trap to study Allophycocyanin (APC), a photosynthetic antenna-protein and popular fluorescent probe. A complex relationship between fluorescence intensity and lifetime is observed, suggesting light-induced conformational changes and radiative and non-radiative rate fluctuations.  Second, we apply the ABEL Trap to single molecules of the multi-copper enzyme blue Nitrite Reductase where a fluorescent label reports on the oxidation state of the Type I Copper.  Redox cycling is observed and kinetic analysis allows extraction of the microscopic rate constants in the kinetic scheme.  Evidence of a substrate-induced shift of the intramolecular electron transfer rate is seen.  Taken together, these observations provide new windows into dynamics of solution-phase fluorescent proteins and enzymes.

 

 

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Achilles Kapanidis

“Conformational dynamics of DNA polymerase”

 

 

The remarkable fidelity of DNA polymerases depends largely on their efficient rejection of incorrect nucleotides prior to nucleotide addition. We use single-molecule FRET to examine fidelity-related conformational transitions preceding nucleotide addition by DNA polymerase I (Klenow fragment). Our experiments distinguished the open and closed conformations that predominate in the binary Pol-DNA and ternary Pol-DNA-dNTP(complementary) complexes, and showed that the unliganded polymerase is highly conformationally flexible. We also showed that ternary complexes with mismatched dNTPs or complementary ribonucleotides form novel FRET species perhaps corresponding to partially closed conformations which may act as kinetic checkpoints crucial for fidelity. We have also studied how amino acids proximal to the polymerase active site contribute to fidelity by examining the conformational states of polymerase derivatives that act as “mutators”, i.e., have decreased fidelity. For example, an E710A substitution (glutamic acid to alanine) reduces the fidelity through a mechanism that remains unclear. Our studies of a doubly labelled E710A polymerase (Pol[E710A]) showed that  the binary complex favors the open conformation more than the wild-type. More intriguingly, addition of the complementary dNTP to a binary Pol[E710A]-DNA complex does not form the closed conformation seen in the ternary complex of the wild-type polymerase; in contrast, and at high nucleotide concentrations, Pol[E710A] adopts a FRET state similar to that in ternary complexes of wild-type polymerase with mismatched dNTPs or complementary ribonucleotides; this state may correspond to a partially closed conformation, and is also adopted by Pol[E710A]-DNA-dNTP(mismatched) complexes, offering a possible explanation for the reduced fidelity associated with E710A. Since Pol[E710A] can perform DNA synthesis, our results raise the question whether polymerisation occurs from a partially closed conformation or a transiently formed closed complex; experiments to address this question are in progress, along with studies of additional mutators.

Hohlbein J, Santoso Y, Joyce CM, Potapova O, Le Reste L, Evans, G., Torella JP, Grindley NDF, and Kapanidis AN

 

 

 

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Rudolph Rigler

“Stochastic Events and Correlated Processes in Single Molecule Catalysis”

 

Substrate product catalysis mediated by single surface bound horse radish peroxidase  leads to stochastic intensity fluctuations of fluorogenic substrates  (Edman et al. 1999). The behavior which is characterized by active and lazy periods of the substrate turnover (Hassler et al. 2005) is linked to a distribution of rates which are characterized by stretched exponentials. Their time dependence relates to the decay of Non Markovian processes representing the lifetime of a structural memory (Edman and Rigler,2000, Hassler et al.2007, Rigler,2010) Detailed models will be discussed.

 

Edman,L. et al. 1999, Chem.Phys. 247,11

Edman.L.& Rigler,R. 2000, PNAS .97,8266      

Hassler,K, et al.  2005, Biophys.J.104,L01

 2007, Optics Express,15,5366

Rigler,R  2010, in Single Molecule Spectroscopy in  Chemistry,  Physics and Biology,Springer

 

 

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Gerhard J. Schütz

"Addressing plasma membrane nanostructures by single molecule techniques"

 

Current scientific research throughout the natural sciences aims at the exploration of the Nanocosm, the collectivity of structures with dimensions between 1 and 100nm. In the life sciences, the diversity of this Nanocosm attracts more and more researchers to the emerging field of Nanobiotechnology. In my lecture, I will show examples how to obtain insights into the organization of the cellular Nanocosm by single molecule experiments.

Our primary goal is an understanding of the role of such structures for immune recognition. For this, we apply single molecule tracking to resolve the plasma membrane structure at sub-diffraction-limited length-scales by employing the high precision for localizing biomolecules of ~15nm (1-5). Brightness and single molecule colocalization analysis allows us to study stable or transient molecular associations in vivo (6). In particular, I will present results on the interaction between antigen-loaded MHC and the T cell receptor directly in the interface region of a T cell with a mimicry of an antigen-presenting cell (7).

1.    Wieser, S., Axmann, M., and Schütz, G. J. (2008) Biophys J 95, 5988-6001

2.    Wieser, S., Moertelmaier, M., Fuertbauer, E., Stockinger, H., and Schütz, G. J. (2007) Biophys J 92, 3719-3728

3.    Wieser, S., and Schütz, G. J. (2008) Methods 46, 131-140

4.    Wieser, S., Schütz, G. J., Cooper, M. E., and Stockinger, H. (2007) Appl Phys Lett 91, 233901

5.    Wieser, S., Weghuber, J., Sams, M., Stockinger, H., and Schütz, G. J. (2009) Soft Matter 5, 3287-3294

6.    Moertelmaier, M., Brameshuber, M., Linimeier, M., Schütz, G. J., and Stockinger, H. (2005) Appl Phys Lett 87, 263903

7.    Huppa, J. B., Axmann, M., Mortelmaier, M. A., Lillemeier, B. F., Newell, E. W., Brameshuber, M., Klein, L. O., Schütz, G. J., and Davis, M. M. (2010) Nature 463, 963-967

 

 

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Herman Spaink

“Perspectives of single enzyme imaging in zebrafish (20 mins) and Discussion session”

 

 

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Leandro C. Tabares

“Picturing the activity of the Cu-containing Nitrite Reductase at the single molecule level”

 

Click here for abstract

 

 

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Antoine M. van Oijen

“Single-molecule studies of multi-protein machines”

 

Novel nanomanipulation and imaging methods have made it possible to study biochemical reactions at the level of individual proteins. In a biological context, most of these proteins function in concert with others in multi-protein complexes, so an important future direction is the utilization of single-molecule techniques to unravel the orchestration of large macromolecular assemblies. I will discuss our single-molecule studies of the replisome, the multi-protein machinery that is responsible for replication of DNA. I will present experiments that rely on the readout of mechanical DNA properties as well as single-molecule fluorescence imaging to obtain information on the catalytic activity and dynamic composition of the replisome.

Zernike Institute for Advanced Materials, Centre for Synthetic Biology, University of Groningen, Nijenborgh 4, 9747 AG   Groningen, a.m.van.oijen@rug.nl

 

 

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Shimon Weiss

Multiscale, Superresolved, Ultrasensitive Optical Molecular Imaging”

 

We will overview the utilization of single molecule and superresolution imaging tools to the study of molecular machines, interactions, and circuits in-vitro, in live cells and in small organisms

 

 

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X. Sunney Xie

"Single molecule insights into enzyme catalysis and allostery"

 

The mechanisms of enzyme catalysis and allostery have been longstanding and fundamental questions in biophysical chemistry.   I will talk about what we have learned on this subject.

Harvard University, Cambridge Ma, USA

 

 

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Haw Yang

“Protein Large-Amplitude Conformational Transitions: Dynamics, Mechanics, and Functional Roles”

 

Many enzymes mould their structures to poise substrates in their active sites for chemical reaction such that conformational remodeling is necessary during each catalytic cycle. In both protein tyrosine phosphatase B, PtpB, from M. tuberculosis (a virulence factor of tuberculosis and a potential drug target) and adenylate kinase, AK, from E. coli (a ubiquitous energy-balancing enzyme in cells), the conformational change involves large-amplitude rearrangements of the enzyme’s lid domain. These domain movements have been followed in real time on their respective catalytic timescales using high-resolution single-molecule Förster resonance energy transfer (FRET) spectroscopy. It is shown quantitatively that both PtpB and AK are capable of dynamically sampling two distinct states that correlate well with those observed by x-ray crystallography. For AK, the experiments generalized Koshland’s concept to “dynamical induced fit,” and showed that interaction with substrates restricts the spatial extent of conformational fluctuations rather than locking the enzyme into a compact state. Integrating these microscopic dynamics into macroscopic kinetics allows us to place the experimentally measured free-energy landscape in the context of enzymatic turnovers. Comparison with molecular dynamics simulations suggests that a structural element underlying the long-term dynamics is partial unfolding at the hinge region. This “local unfolding” concept was supported experimentally in the case of PtpB. If time allows, we will also discuss how single-molecule experiments help to advance ideas in molecular mechanics.



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