Improved interpretation of 14-3-3ζ fluorescence measurements by molecular dynamics simulations

Hana Zigová1, Gabor Nagy2, Chris Oostenbrink3, Jozef Hritz1

1CEITEC MU, Masaryk University, Kamenice 753/5, 625 00 Brno, Czech Republic,

2Max Planck Institute for Biophysical Chemistry, Göttingen, Germany  

3Department of Material Sciences and Process Engineering, Institute for Molecular Modeling and Simulation, University of Natural Resources and Life Sciences, Vienna


Förster resonance energy transfer (FRET) is a spectroscopic method widely used to determine  inter- and intramolecular distances of biomolecules through distance dependent energy transfer between two fluorophores. Apart from distances, the efficiencies of FRET also significantly depend on the mutual orientation of the fluorophores which are generally not accessible to experiments and often crudely assumed to be isotropic. This assumption might become especially invalid for dyes attached to protein surfaces where special interactions can be formed and cause the dye orientations deviate far from isotropic. In order to improve interpretation of FRET measurements, overall dynamics of the dyes attached to the protein can be probed by molecular dynamics (MD) simulation. Obtained ensemble averaged values of FRET efficiency can be directly compared to the experimental values.   

In this study we simulated set of selected donor and acceptor fluorophores connected to the 14-3-3z protein. For that purpose we prepared force-field parameters for the simulated labels compatible with the 54A7 GROMOS force-field. Our MD simulations indicate strong tendency of the dyes to interact with protein surface what significantly restricts the range of possible orientations. However the transitions between potential interaction sites are very slow, what may hinder proper sampling. We note that for two FRET pair variants the donor-acceptor distances and orientation factors differ considerably which results in significant differences in calculated FRET efficiencies. 

The project is financed from the SoMoPro II programme. The research leading to this invention has acquired a financial grant from the People Programme (Marie Curie action) of the Seventh Framework Programme of EU according to the REA Grant Agreement No. 291782. The research is further co-financed by the South-Moravian Region. The article/paper reflects only the author´s views and the Union is not liable for any use that may be made of the information contained therein. In addition, this work was also supported by Czech Science Foundation (15-34684L) and MOBILITY grant CZ 14/2015, WTZ Tschechien 2015-16. The computational simulations were realized in the National Supercomputing Center IT4Innovations, which is supported by The Ministry of Education, Youth and Sports from the Large Infrastructures for Research, Experimental Development and Innovations project „IT4Innovations National Supercomputing Center – LM2015070“. Further computational resources were provided by the MetaCentrum under the program LM2015042 and the CERIT Scientific Cloud under the program LM2015085, provided under the programme "Projects of Large Infrastructure for Research, Development, and Innovations". This research was carried out under the project CEITEC 2020 (LQ1601) with financial support from the Ministry of Education, Youth and Sports of the Czech Republic under the National Sustainability Programme II.