Circular dichroism

Peptides were reconstituted in buffer containing 10 mM sodium phosphate (pH 7.4), and stock solution concentrations were determined using BCA microplates and UV absorption (for full-length peptides). TFE was added at incremental percentages v/v over a range of 0–60%. Each raw data set of absorption had a corresponding buffer scan subtracted from it to remove any absorption contributions from buffer or TFE cosolvent. Resulting data sets were plotted as mean residue ellipticity (MRE) according to the following equation:

where θ is raw ellipticity, MW is the molecular weight of the peptide, N is the number of residues, 1 is the cuvette path length, and c is the peptide concentration in mg/mL. Concentrations of core region peptide samples ranged from 18 to 20 μM for all experiments, whereas full-length IDR peptides were at a concentration of 10 μM. To extract the free energy of folding for Syt 1 IDR peptides, a simultaneous nonlinear least squares fit of 198- and 222-nm wavelengths was performed (mathematical derivation in Supporting Materials and Methods). Four replicate measurements of TFE gradients for each Syt 1 IDR peptide were individually fit and then averaged to determine standard deviations. Estimates of helicity were obtained by fitting data sets to linear combinations of α-helix, β-sheet, and random coil secondary structures as described previously (21, 22).