Fig. 2.

Biophysical characterization of C34coil. (A) CD spectrum of C34, C34-GCN4, and C34coil. The guanidine hydrochloride used to solubilize C34-GCN4 precludes measurements at wavelengths <210 nm. Experimental conditions were: PBS at pH 7.0 at 25°C. C34coil exhibits a helical structure ([θ]₂₂₂ value of –29,400 deg cm²·dmol^–1), whereas C34 (–3,800 deg cm²·dmol^–1) and the C34-GCN4 peptide (–12,400 deg cm²·dmol^–1) exhibit relatively unstructured conformations. (B) Guanidine hydrochloride denaturation of C34coil, as monitored by CD spectroscopy at 222 nm. The fitted curve to a two-state unfolding transition is shown as a black line. C34coil unfolds at a midpoint of 3.6 M guanidine hydrochloride, with a free energy of unfolding of 9.3 kcal/mol. (C) Apparent molecular weight (MW) of C34coil, as determined by gel filtration chromatography. Shown are the elution times of molecular weight standards (▵) and C34coil (•). Also shown is the best-fit line of apparent MW versus elution time from the molecular weight standards (black line). C34coil exhibits an apparent molecular mass of 7,000 ± 2,000 Da (expected molecular mass of 8,740 Da for a monomer), with no detectable aggregation. (D) Sensitivity to proteolytic degradation of C34coil and C34. Peptides are incubated with proteinase K at 37°C, and the reaction products are monitored by reverse-phase HPLC. The relative ratios of proteinase K account for differences in both the protease concentrations and incubation times. Shown are the HPLC chromatograms. C34coil is ≈1,000-fold more resistant to degradation by proteinase K than by C34.