2/1 - Special Seminar (physical): Kerstin Blank, Radboud University Nijmegen, The Netherlands
Kerstin Blank - speaker's webpage
Institute for Molecules and Materials
Radboud University Nijmegen, The Netherlands
Host: Haw Yang
Single Enzyme Kinetics: optical and nanoelectronic approaches
Single molecule experiments are now commonly used for monitoring conformational changes of enzymes. Despite the power of the single molecule approach to also investigate the kinetics of the catalytic reaction itself, the field of single enzyme kinetics is only about to leave the proof-of-principle stage. The ability of accurately recording the sequence of enzymatic turnovers is the crucial starting point for the construction of kinetic schemes . Using the two well-characterized proteases α-chymotrypsin and thermolysin as model enzymes, I will present a fluorescence-based and an electronic approach towards a detailed analysis of their kinetics at the single molecule level.
The fluorescence-based approach utilizes fluorogenic substrates. Every catalytic event releases a fluorophore that is detected with a confocal fluorescence microscope. I will discuss the importance of the substrate design for obtaining accurate kinetic data [2,3] and show the applicability of this approach to investigate the pH dependent regulation of α-chymotrypsin activity. The nanoelectronic approach aims to detect the single enzyme turnover sequence using a carbon nanotube field effect transistor. This sensor reports on the CNT conductance that is influenced by changes in the local electrostatic environment around an immobilized enzyme . After introducing the detection scheme, I will focus on immobilization methods  and highlight first results using the enzyme thermolysin.
 V. I. Claessen, H. Engelkamp, P. C. M Christianen, J. C. Maan, R. J. M. Nolte, K. Blank and A. E. Rowan. (2010) Single-Biomolecule Kinetics: The Art of Studying a Single Enzyme. Annu Rev Anal Chem 3:319
 T. G. Terentyeva, W. Van Rossom, M. Van der Auweraer, K. Blank and J. Hofkens (2011) Morpholinecarbonyl-Rhodamine 110 based Substrates for the Determination of Protease Activity with Accurate Kinetic Parameters. Bioconjugate Chem 22:1932
 T. G. Terentyeva, H. Engelkamp, T. Komatsuzaki, A. E. Rowan, J. Hofkens, C.-B. Li and K. Blank (2012) Dynamic Disorder in Single Enzyme Experiments: Facts and Artifacts. ACS Nano 6:346
 Y. Choi, I. S. Moody, P. C. Sims, S. R. Hunt, B. L. Corso, I. Perez, G. A. Weiss and P. G. Collins (2012) Single-Molecule Lysozyme Dynamics Monitored by an Electronic Circuit. Science 335:319
 L. Prisbrey, D. Roundy, K. Blank, L. S. Fifield and E. D. Minot (2012) Electrical Characteristics Of Carbon Nanotube Devices Prepared with Single Oxidative Point Defects. J Phys Chem C 116:1961