07.04.2020 08:19

New functional states observed in single enzymes

Von: Redaktion / V.M.

Scientists from the Chair of Molecular Physical Chemistry and the Institute of Pharmaceutical and Medicinal Chemistry at Heinrich-Heine-University as well as the Forschungszentrum Jülich together with a team of researchers at Clemson University, Clemson, USA and University of California, Los Angeles, USA developed and demonstrated the use of optical imaging methods to monitor single enzyme molecules in action. The fluorescence-based technique in combination with structural bioinformatics may accelerate the field of structural biology, helping scientists better understand how molecules are assembled, function, and interact, and ultimately may aid in modulating such properties.

Claus Seidel, Hugo Sanabria, and Holger Gohlke, together with their colleagues, used distance dependent Förster resonance energy transfer (FRET) between two specifically attached fluorescent probes to unravel the kinetic and dynamic interplay of the conformational states of the enzyme lysozyme of bacteriophage T4 (T4L). The human homologue is found in tears and mucus. There, the enzyme destroys the protective carbohydrate chains surrounding bacterial cell walls. They reported their findings in the journal Nature Communications.

Claus Seidel said “we underpin essential reaction steps of biomolecular machines (enzymes). Our FRET studies demonstrate that an extension of the famous classical Michaelis-Menten kinetics from two to three functional enzyme states is essential. The Michaelis-Menten description is one of the best-known models of enzyme kinetics“.

The centerpiece of this imaging tool is a FRET-based microscope, a sophisticated and powerful machine capable of visualizing biomolecules as small as a few nanometers in solution. To visualize biomolecules at work, the authors placed two fluorescent markers on a set of molecules resulting in a ruler at the molecular level. By using different locations of the markers, the team collected a set of distances that describe structural states of the observed molecule. In essence, this process generates a collection of data points that, with the help of structural bioinformatics, is used to distinguish how the molecule looks like and how it moves. “We observe changes in the structure, and because our signal is time-dependent, we can also get an idea of how the molecule is moving over time”, Sanabria said. “Together with molecular modeling and simulations, such data can yield state-specific structural information on complex dynamic biomolecular assemblies”, Gohlke added. “Here we tracked T4L as it processes its substrate at near atomistic level with unprecedented spatial and temporal resolution (micro- to milliseconds). We’ve taken the FRET spectroscopy field to a whole new level”, Sanabria concluded.

The work revealed that the structural space of this classical and extensively studied enzyme is actually wider than previously thought. Until now, scientists have largely determined the structures of proteins like lysozyme mainly using methods such as X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryo-electron microscopy. “For the longest time, this molecule was considered a two-state molecule because of how it receives the substrate of the cell wall of the target bacteria,” Sanabria said. “However, we have identified a new third functional state that is essential in the reaction cycle of an enzyme so that an extended Michaelis-Menten Mechanism is needed to describe enzyme catalysis.”

Furthermore, the team is helping to establish a database (https://pdb-dev.wwpdb.org) where their FRET-based structural models of similarly generated biomolecular models can be stored and accessed by other scientists. Together with the FRET community (www.FRET.community) the group is also working to establish recommendations for FRET microscopy.

Claus A.M. Seidel, Chair Molecular Physical Chemistry, Heinrich Heine University Düsseldorf, Germany

cseidel(at)hhu.de

 

Hugo Sanabria, Associate Professor of Physics, Clemson University, Clemson, SC, USA

hsanabr(at)clemson.edu

 

Original publication

Sanabria, H., Rodnin, D., Hemmen, K., Peulen, T.-O., Felekyan, S., Fleissner, M. R., Dimura, M., Koberling, F., Kühnemuth, R., Hubbell, W., Gohlke, H., Seidel, C. A. M., "Resolving dynamics and function of transient states in single enzyme molecules", Nature Communications 11, e1231 (2020)

DOI: hsanabr(at)clemson.edu

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