LAUSR

Chemical cross-linking combined with MALDI-MS was applied to structural analysis of a protein nanocontainer. Specifically, an engineered variant of lumazine synthase from Aquifex aeolicus (AaLS-13) was investigated that self-assembles into a capsid-like structure and is known to encapsulate other proteins by Coulombic attraction. Two complementary soft ionization techniques, MALDI-MS and native ESI-MS, were utilized to map the subunit stoichiometry of the high molecular weight capsid. In accordance with the previously reported cryo-electron microscopy structure of this protein container, only pentameric subunits were detected. This study highlights the possibility to map subunit stoichiometry via chemical cross-linking with glutaraldehyde followed by MALDI-MS. The same approach was used to study protein-protein interactions during encapsulation of GFP(+36) by the AaLS-13 capsid. Heterocomplexes between GFP(+36) and AaLS-13 multimers were not observed when mixed at maximal loading capacity (AalS-13 monomer:GFP(+36) 4:1). This is in agreement with the known fast encapsulation of GFP(+36) by the protein capsid, which essentially removes any free GFP(+36) from the solution. Exceeding the maximal loading capacity by addition of excess GFP(+36) results in aggregation. This article is protected by copyright. All rights reserved.