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

 

Scientific Report

 

One of the exciting developments in single-molecule spectroscopy is the study of enzyme activity and turn-over at the single-molecule level. 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. This workshop addressed the challenge of taking the study of single enzyme catalysis beyond the proof-of-principle stage, bringing together leading players in this field.

 

What do we know? Xie and others have found that a single enzyme molecule exhibits fluctuations of catalytic rates over a wide range of time scales, i.e., a single enzyme molecule does not have a rate constant! Rigler, Hammes and Xie discussed the general mechanism that is beginning to emerge. It is a multiple-pathway, multiple intermediate scheme involving conformational ensembles.  This mechanism has to be be described in terms of transition state theory and free energy surfaces.

 

Many experiments corroborate these observations. Yet in the discussion it also was pointed that  developments in this field are hampered by a number of constraints. For example, the necessity of non-native, fluorogenic substrates is a severely limiting factor, and may not be representative for the natural catalytic process. Immobilization of the enzyme is required, but may affect the range and dynamics of conformational fluctuations. Finally, brighter probes are needed with improved photostability. Potential breakthroughs presented at the meeting are highlighted below.

 

FRET-based optical and electrochemical studies of redox enzymes down to the single-molecule level: A new method was presented by Canters, Aartsma and co-workers for monitoring redox enzyme activity at the single-molecule level. It is applicable to a large variety of enzyme systems and does not interfere with the enzymes’ natural activity. Feasibility was demonstrated of trapping enzymes in the Anti-Brownian Electrokinetic trap (ABEL trap), developed by Moerner and co-workers and discussed by Goldsmith, eliminating the Brownian motion.  It is a promising alternative for immobilization schemes. Dual focus fluorescence correlation spectroscopy (FCS) was described by Enderlein, which allows for more precise and quantitative measurements of molecular dynamics and diffusion parameters of biomolecules. An important future direction is the utilization of single-molecule techniques to unravel the orchestration of large macromolecular assemblies. Van Oijen discussed single-molecule studies of the replisome, the multi-protein machinery that is responsible for replication of DNA. J. Cao presented and discussed a generalized form of the Michaelis-Menten (MM) equation, providing a unified approach to analyze non-MM behavior and to describe cooperativity, inhibition, and multi-stability. The potential of nanoparticles as optical probes and for monitoring catalysis was discussed by Orrit and Chen. The most important feature is the great photostability of nanoparticles. The cellular and biological contexts of single-enzyme experiments were discussed by Schmidt, Schütz, and Spaink.

 

The future of single-molecule imaging and spectroscopy calls for higher throughput and better temporal resolution. Important technological advances were presented by Weiss.  He described superresolution optical fluctuation imaging (SOFI), a new 3D super-resolution method based on the analysis of temporal fluorescence fluctuations of emitters that works with any wide-field microscope. The enhanced resolution results in striking images of cellular structures. Weiss also reported on the development of two new devices consisting of arrays of single-photon detectors. In combination with the possibility to generate multiple focal spots from a single laser beam, a system for parallel data acquisition can be constructed with greatly improved throughput.  These detectors are still largely experimental, but they mark the direction in which the field is developing.

 

The workshop has resulted in an agreement between Moerner, Canters and Aartsma to collaborate on combining the ABEL trap with single-enzyme studies. Preliminary data had been obtained, but they will seek financial funding to establish a solid footing for this research. A number of participants, headed by L.J.C. Jeuken, have submitted a joint proposal for an FP7/ITN network as a follow-up to this workshop.

 

T.J. Aartsma

 



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