Thermostability of protein nanocages: the effect of natural extra peptide on the exterior surface

Abstract

Protein nanocages have been used as functional bio-templates for the synthesis or organization of nanomaterials. However, the stability of these protein nanocages is nonideal, which limits their applications. Herein, we characterized the high thermal stability of plant ferritin, soybean seed H-2 ferritin (SSFH-2), the melting point (T _m) of which is 106 °C. We demonstrated that the hyperthermostability of SSFH-2 is derived from extra peptides (EP) located on its outer surface. Indeed, removal of the EP domains resulted in a dramatic decrease in T _m to 88 °C. Similar to EP-deleted plant ferritin, human H-chain ferritin (HuHF) has a T _m of 82 °C. Excitingly, the graft of the EP domain on the exterior surface of HuHF pronouncedly improved its T _m to 103 °C, which represents a simple, efficient approach to the construction of protein architectures with high stability. The remarkable stability of protein nanocages will greatly facilitate their application as robust biotemplates in the field of nanoscience.

Fig. 1. Schematic representation of reversible control of the hyperthermostability of ferritin nanocage by extra peptide (EP). With EP domain located on its outer surface, ferritin exhibits hyperthermostability (value of T_m above 100 °C).

Fig. 2. Characterization of the structure of SSFH-2 after thermal treatment at 100 °C for different times. (a) SDS-PAGE and (b) native PAGE analyses of thermal-treated SSFH-2. Lane M, protein markers and their corresponding molecular masses; lanes 1, 2, 3, 4, and 5 correspond to SSFH-2 upon treated at 100 °C for 0, 5, 10, 20, and 30 min, respectively. (c) CD spectra and (d) Fluorescence spectra of SSFH-2 upon thermal treatment for 5, 10, 20, and 30 min. (e) TEM image of SSFH-2 at 100 °C for 30 min. Scale bar represent 100 nm. (f) Dynamic light scattering (DLS) analyses of untreated SSFH-2 and upon thermal treatment at 100 °C for 30 min. Conditions: 1.0 μM SSFH-2 in 50 mM MOPS, pH 7.9.

Fig. 3. The structural basis of the hyperthermostability of SSFH-2. (a) DSC curves of SSFH-2 and its EP-deleted mutant SSFH-2-ΔEP, at scan rate of 10 °C min⁻¹. (b) The thermal stability of SSFH-2 as a function of treatment time at fixed temperature 100 °C. (c) The crystal structure of SSFH-2 upon treated at 100 °C for 5 min, which is viewed down the 4-fold channels (PDB ID: 6J4M). (d) The position of newly formed noncovalent interactions upon thermal treatment. (e–g) Superposition of untreated (pink) and thermal-treated (blue) SSFH-2 subunit revealed the formation of several new types of new noncovalent interactions upon treated at 100 °C for 5 min.

Fig. 4. The crystal structure and thermal stability of HuHF and EP-inserted mutant (HuHF-∇EP). (a) The crystal structure of HuHF-∇EP viewed down the 4-fold channels (PDB ID: 6J4A). (b) The body-centered-cubic (BCC) packing arrangement of HuHF-∇EP crystals. (c and d) DSC curves of native HuHF (c) and its mutant HuHF-∇EP (d), at scan rate of 10 °C min⁻¹.