C-terminal segment of yeast BMH proteins exhibits different structure compared to other 14-3-3 protein isoforms

D. Veisova¹, L. Rezabkova^1,2, P. Herman³, J. Vecer³, T. Obsil^1,2, V. Obsilova¹

¹Institute of Physiology Academy of Sciences of the Czech Republic, 14220 Prague, Czech Republic

²Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, 12843 Prague, Czech Republic

³Faculty of Mathematics and Physics, Institute of Physics, Charles University, 12116 Prague, Czech Republic

obsilova@biomed.cas.cz

Yeast 14-3-3 protein isoforms BMH1 and BMH2 posses a distinctly variant C-terminal tail which differs them from the isoforms of higher eukaryotes. Their C-termini are longer and contains polyglutamine stretch of unknown function. It is now well established that the C-terminal segment of 14-3-3 proteins plays an important regulatory role by functioning as an autoinhibitor which can occupy the ligand binding groove and blocks the binding of inappropriate ligands [1-3]. Whether the same holds true or not for the yeast isoforms is unclear. Therefore, we investigated the conformational behavior of the C-terminal segment of BMH proteins using various biophysical techniques. Dynamic light scattering, time-resolved fluorescence anisotropy decay and size exclusion chromatography measurements showed that the molecules of BMH proteins are significantly bigger compared to the human 14-3-3 zeta isoform. On the other hand, the sedimentation equilibrium analysis confirmed that BMH proteins form dimers. Time-resolved tryptophan fluorescence experiments revealed no dramatic structural changes of the C-terminal segment upon the ligand binding. Taken together, the C-terminal segment of BMH proteins adopts very open and extended conformation that increases their apparent molecular size. It seems, therefore, that the C-terminal segment of BMH proteins does not function as an autoinhibitor and does not block their ligand binding grooves.

References

1. A.B. Truong, S.C. Masters, H. Yang, H. Fu, Proteins, 49, (2002), 321.

2. V. Obsilova, P. Herman, J. Vecer, M. sulc, J. Teisinger, T. Obsil, J. Biol. Chem., 279, (2004) 4531.

3. J. Silhan, V. Obsilova, J. Vecer, P. Herman, M. Sulc, J. teisinger, T. Obsil, J. Biol. Chem., 279, (2004), 49113.

Acknowledgements.

This work was funded by Grant IAA501110801 of the Grant Agency of the Academy of Sciences of the Czech Republic, by Research Project MSM0021620857 and Centre of Neurosciences LC554 of the Ministry of Education, Youth, and Sports of the Czech Republic, and by Research Project AV0Z50110509 of the Academy of Sciences of the Czech Republic.