Protein symmetrization as a novel tool in structural biology

Orateur : Soutenance de Thèse de Franscesca COSCIA (IBS/VIC)

Recent technological advances have allowed de novo protein structural determination from cryoEM maps. Although a highly desirable alternative to X-ray crystallography, whose bottleneck is to grow well diffracting crystals, cryoEM is not suited to most monomeric biomedical relevant proteins, that are below its size limit ( 100 kDa) and lack of symmetry. Our idea is genetically fusing a target protein of interest to with a homo-oligomeric template protein, thereby increasing its size and symmetry and making possible its analysis by cryoEM. To demonstrate proof-of-principle of protein symmetrization we designed and ranked by several biophysical techniques and negative stain EM different combinations between known targets and two different templates with D6 and icosahedral symmetry. Hence, we identified a promising dodecameric chimera for structural analysis, comprising maltose binding protein (MBP) connected to Glutamine synthetase (GS). The 10 Å resolution electron density of this symmetrized 40 kDa MBP presents shape and features corresponding to the known atomic structure. This result establishes the proof-of-concept that protein symmetrization can be used for the structure determination of monomeric proteins below 100 kDa by cryoEM, thereby providing a promising new tool for analyzing targets resistant to conventional structural analysis.

Télécharger dans mon agenda