Backbone circularization of protein is a powerful method to improve its structural stability. In this paper, we presumed that a tight connection leads to much higher stability. Therefore, we designed circularized variants of a granulocyte-colony stimulating factor (G-CSF) with a structurally optimized terminal connection. To estimate the appropriate length of the connection, we surveyed the Protein Data Bank to find local structures as a model for the connecting segment. We set the library of local structures composed of “helix–loop–helix,” subsequently selected entries similar to the G-CSF terminus, and finally sorted the hit structures according to the loop length. Two, five, or nine loop residues were frequently observed; thus, three circularized variants (C163, C166, and C170) were constructed, prepared, and evaluated. All circularized variants demonstrated a higher thermal stability than linear G-CSF (L175). In particular, C166 that retained five connecting residues demonstrated apparent T m values of 69.4 °C, which is 8.7 °C higher than that of the circularized variant with no truncation (C177), indicating that the optimization of the connecting segment is effective for enhancing the overall structural stability. C166 also showed higher proteolytic stability against both endoprotease and exopeptidase than L175. We anticipate that the present study will contribute to the improvement in the general design of circularized protein and development of G-CSF biobetters.

The findings may result from poor shelter or dwelling conditions, as well as overpopulation and lack of basic resources. Adequate shelters, supply systems, and protection from infection, including vaccinations, are needed to prevent deteriorations in health status after the acute phase of a natural disaster.

Monoclonal immunoglobulin G (IgG) is a multidomain protein. It has been reported that the conformational and colloidal stabilities of each domain are different, and it is predicted that limited domains participate in IgG aggregation. In contrast, the influence of interdomain interactions on IgG aggregation remains unclear. The fragment crystallizable (Fc) region is also a multidomain protein consisting of two sets of C2 and C3 domains. Here, we have analyzed the conformational change and aggregate size of an aglycosylated Fc region induced by both acid and salt stresses and have elucidated the influence of interdomain interactions between C2 and C3 domains on the conformational and colloidal stabilities of the aglycosylated Fc region. Singular value decomposition analyses demonstrated that the C2 and C3 domains unfolded almost independently from each other in the aglycosylated Fc region. Meanwhile, the colloidal stabilities of the C2 and C3 domains affect the aggregation process of the unfolded aglycosylated Fc region in a compensatory way. Moreover, the influence of an additional interdomain disulfide bond, introduced at the C-terminal end of the C3 domains to produce the Fc variant, cyclized Fc, was evaluated. This interdomain disulfide bond increased the conformational stability of the C3 domain. The stabilization of the C3 domain in the cyclized Fc successfully improved aggregation tolerance following acid stress, although the sizes of aggregates produced were comparable to those of the aglycosylated Fc region.

The N-linked glycan in immunoglobulin G is critical for the stability and function of the crystallizable fragment (Fc) region. Alteration of these protein properties upon the removal of the N-linked glycan has often been explained by the alteration of the C H 2 domain orientation in the Fc region. To confirm this hypothesis, we examined the small-angle X-ray scattering (SAXS) profile of the glycosylated Fc region (gFc) and aglycosylated Fc region (aFc) in solution. Conformational characteristics of the C H 2 domain orientation were validated by comparison with SAXS profiles theoretically calculated from multiple crystal structures of the Fc region with different C H 2 domain orientations. The reduced chi-square values from the fitting analyses of gFc and aFc associated with the degree of openness or closure of each crystal structure, as determined from the first principal component that partially governed the variation of the C H 2 domain orientation extracted by a singular value decomposition analysis. For both gFc and aFc, the best-fitted SAXS profiles corresponded to ones calculated based on the crystal structure of gFc that formed a "semi-closed" C H 2 domain orientation. Collectively, the data indicated that the removal of the N-linked glycan only negligibly affected the C H 2 domain orientation in solution. These findings will guide the development of methodology for the production of highly refined functional Fc variants.