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Protein symmetrization as a novel tool in structural biology

Abstract : Structural determination of proteins at atomic level resolution is crucial for unravelling their function. X-ray crystallography has successfully been used to determine macromolecular structures with sizes ranging from kDa to MDa, and currently remains the most efficient method for the high-resolution structure determination of monomeric proteins within the 40-100 kDa range. However, this method is limited by the ability to grow well diffracting crystals, which is problematic for several targets, such as membrane proteins. Single particle cryo electron microscopy (cryoEM) allows near atomic (3-4Å) resolution structural determination of large, preferably symmetric, assemblies in solution. Biological molecules scatter electrons weakly and, to avoid radiation damage, only low electron doses can be used during imaging. Consequently, raw cryoEM images are extremely noisy. However, averaging many molecular images aligned in the same orientation permits one to increase the signal-to-noise ratio, ultimately allowing the achievement of a 3D density map of the molecule of interest. Nevertheless, as the molecular size and degree of symmetry decrease, the individual images loose adequate features for accurate alignment. Currently, cryoEM analysis is practically impossible for monomeric proteins below ~100 kDa in mass. We propose to circumvent this obstacle by fusing such monomeric target proteins to a homo-oligomeric protein (template), thereby generating a self-assembling particle whose large size and symmetry should facilitate cryoEM analysis. In the present thesis we seek to test and demonstrate the feasibility of this ‘protein symmetrization' approach and to evaluate its usefulness for protein structure determination. To set up the pilot study we combined selected targets of known structure with two templates: Glutamine Synthetase (GS), a 12-mer with D6 symmetry and a helical N-terminus, and the E2 subunit of the pyruvate dehydrogenase complex, a 60-mer with icosahedral symmetry and an unstructured N-terminus. After recombinant production in E.coli we identified by negative stain EM a promising dodecameric chimera for structural analysis, comprising maltose binding protein (Mbp) connected to GS by a tri-alanine linker (denoted “Mag”). In order to optimize sample homogeneity we produced a panel of Mag deletion constructs by sequentially truncating the 17 residues between the Mbp and GS domains. A combination of biophysical techniques (thermal shift assay, dynamic light scattering, size exclusion chromatography) and negative stain EM allowed us to select the best candidate for cryoEM analysis, MagΔ5. By enforcing D6 symmetry we obtained a cryoEM map with a resolution of 10Å (FSC 0.5 criterion). The density of the symmetrized 40 kDa Mbp presents shape and features corresponding to the known atomic structure. In particular, the catalytic pocket and specific α-helical elements are distinguishable. The cryoEM map is additionally validated by a 7Å crystal structure of the MagΔ5 oligomer. The presence of a continuous helical connection between target (Mbp) and template (GS) likely contributed to the conformational homogeneity of MagΔ5. Moreover, comparing MagΔ5 with other chimeras studied in this work suggests that a large buried surface area and favorable interactions between the target and template limit the flexibility of the chimera and improve its resolution by cryoEM. For the symmetrization of a target of unknown structure, we envisage proceeding by a trial and error approach by fusing it to a panel of templates with helical termini and different surface properties, and subsequently selecting the best ones using biophysical assays. In conclusion, the present work 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.
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Submitted on : Tuesday, July 12, 2016 - 12:19:07 PM
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  • HAL Id : tel-01344632, version 1



Francesca Coscia. Protein symmetrization as a novel tool in structural biology. Biomolecules [q-bio.BM]. Université de Grenoble, 2014. English. ⟨NNT : 2014GRENV066⟩. ⟨tel-01344632⟩



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