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.