Backbone Circularization Coupled with Optimization of Connecting Segment in Effectively Improving the Stability of Granulocyte-Colony Stimulating Factor

Abstract

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.

Stabilization of backbone‐circularized protein is attained by synergistic gains in enthalpy of folded structure and entropy of unfolded structure

“…). This was consistent with the order of the apparent T m values determined by evaluating the loss of secondary structure with increasing temperatures . The apparent T m includes information about reversible unfolding and subsequent irreversible denaturation (i.e., aggregation).…”

“…Cysteine bridge reduction by the addition of dithiothreitol led to a 4‐Da increase in the molecular mass of all variants, indicating that two disulfide bonds were formed in each variant. In the previous study , we had shown that the native G‐CSF and G‐CSF variants (used in the current study) had similar binding affinities to the G‐CSF receptor. This suggested that these variants retained the original conformation of the native G‐CSF including the native connectivity of the two disulfide bonds.…”

“…The thermodynamic stability showed the same trend as heat tolerance, as C M,IU values of C166, C177, and L175 were 2.8, 2.6, and 2.0 m , respectively. Comparing heat tolerance and chemical denaturation at pH 4.0 with thermal denaturation at pH 8.0 (determined in a previous study ) demonstrated that the relative order of conformational stability among the variants is not pH‐dependent. Therefore, the conformational stability at physiological pH can be qualitatively evaluated by examining the differences in conformational stability at pH 4.…”

“…The circularized G‐CSF variants characterized in the current study were developed prior to the study . When initially designing them, we hypothesized that the conformational stability of G‐CSF could be effectively increased by optimizing so that the connector region of the circularized G‐CSF variants becomes tight and distortion‐free .…”

“…Previously, we showed that G‐CSF is amenable to conformational stabilization by backbone circularization as it is a four‐helix bundle protein with adjacent terminal ends . All circularized G‐CSF variants, harboring an engineered segment (connector) of different lengths that links two terminal ends, exhibited superior thermal stability and protease resistance compared to the linear G‐CSF, while maintaining the original function . However, the conformational stability gains associated with backbone circularization differed widely for the circularized variants.…”