Figure 2. NMR spectroscopic characterization of C_L induced C_H1 folding.

(A) ¹⁵N-¹H HSQC spectra of the isolated C_H1 domain (red) and the assigned C_H1 domain in complex with the C_L domain (blue) are shown. In order to characterize the folding pathway of the intrinsically disordered C_H1 domain, time dependent HSQC intensities upon addition of unlabeled C_L to ¹⁵N labeled C_H1 were measured for each assigned residue at the native chemical shift position and fitted by a single exponential function. Two representative traces for Val68 (red) and Lys90 (blue) are shown in the inset. (B) Initial amplitudes for each assigned C_H1 residue were derived from the fitted exponential functions. Residues with an initial HSQC amplitude below a threshold of 25% native intensity are colored in blue and residues above the threshold in red (open bars: residues in loop regions / filled bars: residues in structured regions). Errors indicate standard deviations from single exponential fits. In (C) C_H1 residues with intensities above the threshold in the intermediate are mapped on the crystal structure of the C_H1/C_L dimer (pdb code 1Q9K). The dimerization interface between C_H1 (grey) and C_L (blue) is shown on the left with only residues of C_H1 indicated that are involved in this interaction and above the HSQC amplitude threshold. On the right, internal C_H1 residues above the 25% threshold are shown in red, the three cis proline residues in C_H1 are depicted and labeled in blue. (D) The HSQC spectra of the C_H1 Pro32Ala, Pro34Ala and Pro74Ala mutants show, that only the Pro32Ala mutant is not able to fold in the presence of C_L anymore (blue spectrum). For the other two mutants, Pro34Ala and Pro74Ala, well dispersed spectra and hence folding are observed in the presence of C_L (blue spectra). In the absence of C_L (red spectra), all three mutants show typical HSQC spectra of unfolded proteins. All measurements were carried out in PBS at 25°C except for the folding kinetics which where recorded at 12.5°C.