High‐Mass MALDI‐MS Analysis for the Investigation of Protein Encapsulation within an Engineered Capsid Forming Protein

Abstract

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.