SEC-MALLS-RI
Françoise
Bonneté
RESEARCHER
Alexandre
Pozza
Research Engineer
INTRODUCTION
SEC-MALLS-RI, the coupling of HPLC system with a Size Exclusion Chromatography on line with both Multi-Angle Laser Light Scattering and differential refractometer, is a powerful technique to analyze macromolecules in solution in terms of absolute molar mass, size, oligomeric state, multi-component interactions (e.g. protein-protein, protein-surfactants, protein-ARN). This is an essential tool to evaluate the monodispersity of purified samples prior to low-resolution structural studies (SAXS, SANS) or high-resolution structural studies (Crystallography, Cryo-EM).
Structural studies on membrane proteins (MPs) remain a challenge in biology and pharmaceutical developments, whatever the technique used, NMR, Crystallography or Cryo-EM. In crystallography for example, MP crystallization in detergent micelles (in surfo crystallization), the most commonly used technique, depends on the ability of the detergent to efficiently cover the hydrophobic protein transmembrane domain and avoid possible destabilization and/or aggregation of the protein, while allowing contacts between the polar parts of the MP complex to make crystal growth possible. The choice of detergents or buffer solutions capable of both stabilizing the protein in a functional and stable state and allowing its crystallization is therefore difficult because it is often based on trial-and-error methods. There are few fundamental studies on the characterization/modeling of membrane protein-amphiphile complexes. A thorough knowledge of the structure and dynamics of the amphiphiles associated with MPs and their influence of MP 3D-structure, should allow a better control of sample preparations for high resolution structural studies to elucidate their structure/function relationship.
Depending on the type of detergent used (tail length, head polarity, head volume), or nature of solvent, MPs can bind different amounts of detergent molecules on their transmembrane domain, which can alter their folding, stability and capacity to form crystals or monodisperse complexes.
ShuA in different detergents
By using ShuA from Shigella dysenteriae, a TonB-dependent haem outer membrane transporter (TBDT), as a model system, both its oligomeric state and detergent belt were described in solubilizing detergent (e.g. OPOE) and in commonly used crystallizing detergents (e.g. DDM, OG).
The detergent that forms the smallest belt around ShuA, i.e. OG (Fig 1; Table 1), and gives a monodisperse complex, is that which allowed to crystallize ShuA and to solve its structure by X-Ray crystallography [1] (pdb code: 3FHH).
Finally, using on the one-hand the SEC-MALLS analysis (i.e. the number of detergents bound to ShuA) and on the other-hand, molecular dynamics simulation in a pre-assembled mode (i.e. the 3D-structure of ShuA with the number of detergents bound from SEC-MALLS), a schematic view (Fig 2) of the protein surrounded by a corona of detergents (DDM and OG) as well as interactions of detergent molecules with specific regions of the protein could be described [2].
TSPO in different buffer solutions
Depending on the head group charge or hydration capacity of the amphiphile used to solubilize or purify a membrane protein, the solvent composition (e.g. ionic strength, pH) can modify the shape of the belt around the transmembrane domain of a MP by altering the packing parameter of the amphiphile.
SDS (sodium dodecyl sulfate) is an anionic detergent, which can be used to solubilize membrane proteins from biological membranes or from inclusion bodies. mTSPO from Mus musculus, is a translocator protein, whose the recombinant form is expressed in E. coli mainly in inclusion bodies, and is then solubilized and purified with SDS [3]. In order to prepare suitable samples of mTSPO in SDS for biophysical studies (e.g. SAXS, SANS) [4], different buffer solutions were studied using SEC-MALLS (Fig 3) by varying the ionic strength of salt. If the SDS belt around TSPO increases in size with NaCl, the protein remains monomeric and the complex monodisperse. Coulombic repulsions between TSPO-SDS complexes decrease as observed by the increase in the elution volume and confirmed by SEC-SAXS analysis (Fig 4).
Collaborators
Stéphane Abel (I2BC CEA Saclay) and Karl Brillet (IBMC Strasbourg) for ShuA.
Jean-Jacques Lacapère (LBM, Sorbonne Université) and Sophie Combet (LLB CEA Saclay) for TSPO.
References
1. Brillet, K., Meksem, A., Thompson, A., and Cobessi, D., (2009).Expression, purification, crystallization and preliminary X-ray diffraction analysis of the TonB-dependent haem outer membrane transporter ShuA from Shigella dysenteriae Acta Crystallographica F, https://doi.org/10.1107/S1744309109008148
2. Abel, S., Marchi, M., Solier, J., Finet, S., Brillet, K., and Bonneté, F., (2021).Structural insights into the membrane receptor ShuA in DDM micelles and in a model of gram-negative bacteria outer membrane as seen by SAXS and MD simulations BBA Biomembrane, https://doi.org/10.1016/j.bbamem.2020.183504
3. Robert, J.C. and Lacapère, J.J., (2010).Bacterial overexpressed membrane proteins: an example: the TSPO Methods Mol Biol, 10.1007/978-1-60761-762-4_3
4. Combet, S., Bonneté, F., Finet, S., Pozza, A., Saade, C., Martel, A., Koutsioubas, A., and Lacapère, J.-J., (2023).Effect of amphiphilic environment on the solution structure of mouse TSPO translocator protein Biochimie, https://doi.org/10.1016/j.biochi.2022.11.014