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. 2014 Apr 16;289(22):15666–15679. doi: 10.1074/jbc.M113.534222

FIGURE 8.

FIGURE 8.

Effects of mutations of anti-hCG VHH on its biophysical characteristics. A, CD spectra of wild-type and N52S/N74S/N84T VHH (25 μm) at 37 and 90 °C in HBS-EP buffer without EDTA. B, equilibrium thermal unfolding curves of wild-type and N52S/N74S/N84T VHH were measured by changes in ellipticity at 235 nm at a protein concentration of 25 μm. Curves fitted by standard thermodynamic equations (63) are presented as solid lines, and Tm values of wild-type VHH and N52S/N74S/N84T mutant VHH were estimated to be 65 and 68 °C, respectively. C, Tm values and residual activities of wild-type VHH and VHH mutants (100 nm) after 40 heating-cooling cycles. The Tm values of disulfide mutants C22W/A49C/I70C/C96A (56 °C) and A49C/I70C (74 °C) are from Hagihara et al. (33). Wild-type and N52S/N74S/N84T mutant anti-hCG VHH are abbreviated as WT and Mutant, respectively. Error bars represent S.D. deg, degrees.