Encapsulins are protein nanocompartments that house various cargo enzymes, including a family of decameric ferritin-like proteins. Here, we study a recombinant Haliangium ochraceum encapsulin:encapsulated ferritin complex using cryo–electron microscopy and hydrogen/deuterium exchange mass spectrometry to gain insight into the structural relationship between the encapsulin shell and its protein cargo. An asymmetric single-particle reconstruction reveals four encapsulated ferritin decamers in a tetrahedral arrangement within the encapsulin nanocompartment. This leads to a symmetry mismatch between the protein cargo and the icosahedral encapsulin shell. The encapsulated ferritin decamers are offset from the interior face of the encapsulin shell. Using hydrogen/deuterium exchange mass spectrometry, we observed the dynamic behavior of the major fivefold pore in the encapsulin shell and show the pore opening via the movement of the encapsulin A-domain. These data will accelerate efforts to engineer the encapsulation of heterologous cargo proteins and to alter the permeability of the encapsulin shell via pore modifications.

Encapsulins (Enc) are protein nanocompartments which house various cargo enzymes, including a family of decameric ferritin-like proteins. Previously, we elucidated the structure and activity of these ferritin-like proteins (EncFtn) and demonstrated that they must be encapsulated in a nanocompartment for iron storage. Here, we study a recombinant Haliangium ochraceum Enc:EncFtn complex using electron cryo-microscopy (Cryo-EM) and hydrogen/deuterium exchange mass spectrometry (HDX-MS) to gain insight into the structural relationship between Enc and EncFtn. An asymmetric single particle reconstruction reveals four EncFtn decamers in a tetrahedral arrangement within the encapsulin nanocompartment. This leads to a symmetry mismatch between the EncFtn cargo and the icosahedral encapsulin shell. The EncFtn decamers are offset from the interior face of the encapsulin shell and are resolved at a much lower overall resolution in the final reconstruction. This flexibility, and the fixed number of EncFtn decamers sequestered within, implies that the loading of the encapsulin nanocompartment is limited by the steric effect of both engaged and free encapsulin localization sequences. Using a combination of focused refinements and HDX-MS, we observed dynamic behavior of the major five-fold pore, and show the pore opening via the movement of the encapsulin A-domain. These data can accelerate efforts to engineer the sequestration of heterologous cargo proteins and to alter the permeability of the encapsulin shell via pore modifications.

Encapsulated ferritins assemble into an annular decamer structure via an iron-dependent assembly pathway where dimers associate to form the ferroxidase active site.

Protein glycosylation is one of the most common PTMs and many cell surface receptors, extracellular proteins and biopharmaceuticals are glycosylated. However, HDX-MS analysis of such important glycoproteins has so far been limited by difficulties in determining the HDX of the protein segments that contain glycans. We have developed a column containing immobilized PNGase Rc (from Rudaea cellulosilytica) that can readily be implemented into a conventional HDX-MS setup to allow improved analysis of glycoproteins. We show that HDX-MS with the PNGase Rc column enables efficient online removal of N-linked glycans and the determination of the HDX of glycosylated regions in several complex glycoproteins. Additionally, we use the PNGase Rc column to perform a comprehensive HDX-MS mapping of the binding epitope of a mAb to c-Met, a complex glycoprotein drug target. Importantly, the column retains high activity in the presence of common quench-buffer additives like TCEP and urea and performed consistent across 114 days of extensive use. Overall, our work shows that HDX-MS with the integrated PNGase Rc column can enable fast and efficient online deglycosylation at harsh quench conditions to provide comprehensive analysis of complex glycoproteins.

An atomic absorption Spectrophotometric (AAS) method for determining chelated iron in iron chelate concentrates was collaboratively studied by 13 laboratories. Nonchelated iron was selectively precipitated as ferric hydroxide from an aqueous solution of the sample by adjusting to pH 8.5 with sodium hydroxide. A filtered portion of the sample solution was diluted with 0.5N HC1, and the iron was determined by AAS using standards containing disodium EDTA. Six pairs of iron chelate materials (12 samples) were analyzed and the average coefficients of variation were iron EDTA 3.52%, iron HEDTA 4.16%, iron DTPA 3.55%, iron EDDHA 3.52%, iron citrate 2.86%, and iron DPS 8.47%. The method has been adopted official first action.