Lecture Johan Hofkens
|17 May 2005||FWN-Building 5116.0107, Nijenborgh 4, 9747 AG, Groningen|
|Speaker:||Dr. Johan Hofkens|
|Affiliation:||Department of Chemistry, KULeuven and CMAT, Department of Chemistry, UCL, Belgium|
|Title:||Probing dynamics of individual (bio)molecules by single-molecule spectroscopy|
|Date:||Tue May 17, 2005|
|Telephone:||+31 50 363 4369|
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, three different examples will be presented that highlight the power of single molecule spectroscopy for unraveling hidden heterogeneity and dynamical changes.
Single molecule enzymatics
The first system that will by presented is an enzyme system. 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 hydrophobic cover glass) and a robust enzyme (lipase CalB). With this approach, we were able to monitor single enzyme turnovers for extended periods of time (hours). In order to study the catalysis by a single CALB enzyme, we used as fluorogenic substrate a non-fluorescent ester. In this way, 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. Each active conformation contributes to the overall enzyme turnover cycle (ETOC) a specific exponential factor, an observation, which is often masked by ensemble measurements .
Photo-switching of individual fluorescent proteins
In a last part, the results on the photoswitching in the individual GFP-like fluorescent protein Dronpa from a coral Montipora will be discussed. Ensemble spectroscopy shows that the protonated and the deprotonated form of the chromophore of Dronpa can be reversibly photoswitched on irradiation with a 488 nm and 405 nm light. While the deprotonated form is very bright, the protonated form is almost non-fluorescent due to the fast non-radiative deactivation. Single-molecule two-color excitation measurement shows that the molecule is driven into the protonated form very efficiently when the 405 nm light is absent [2,3]. The fluorescence signal is perfectly recovered on irradiation with the 405 nm light, demonstrating the reversible photoswitching. At the single molecule level, more than 100 switching events could be realized. Our data demonstrate that the response time of the molecule to the light can reach in the order of milliseconds with proper conditions of irradiation. This feature is promising to follow intracellular dynamics in real time.
Polymers at different conditions have been studied, by doping them with probe molecules. Individual molecules reveal the inhomogeneity and complexity of polymers at nanometer scales. While earlier single-molecule experiments focused on the dissolved molecules more recent experiments used the dissolved molecule to probe the polymer. Rotational diffusion dynamics close to the glass transition temperature have been studied by a technique called defocused wide field imaging. In this technique the exact 3D orientation of a molecule can be determined from its emission pattern. We were able to show large dispersion of diffusion parameters from molecule to molecule and as a function of time for one molecule, which is the main source of the well-known non-exponential ensemble kinetics in polymers.
1. Kelly Velonia, Ophir Flomenbom, Davey Loos, Sadahiro Masuo, Mircea Cotlet, Yves Engelborghs, Johan Hofkens, Alan E. Rowan, Joseph Klafter, Roeland, J. M. Nolte, and Frans C. De Schryver,
Single Enzyme kinetics of CALB Catalyzed Hydrolysis, Angewandte Chemie International Edition, 44, 560-564, (2005)
2. Satoshi Habuchi, Mircea Cotlet, Roel Gronheid, Gunter Dirix, Jan Michiels, Jos Vanderleyden, Frans C. De Schryver, J. Hofkens Single-molecule surface enhanced resonance Raman spectroscopy of the enhanced green fluorescent protein J. Am. Chem. Soc. 125 (28), 8446-8447 (2003)
3. Satoshi Habuchi, Ryoko Ando, Peter Dedecker, Wendy Verheijen, Hideaki Mizuno, Atsushi Miyawaki and Johan Hofkens, Reversible single molecule photoswitching in the GFP-like fluorescent protein Dronpa, in press in Proc. Nat. Acad. Sci. USA
|Last modified:||22 October 2012 2.31 p.m.|