Figure 4: Cysteine shotgun labelling experiments.
From: Forced protein unfolding leads to highly elastic and tough protein hydrogels
(a,b). SDS–PAGE shows that Cys23 is buried in the folded state and becomes exposed only after FL-M23C is denatured. a is under ultraviolet illumination and b is under white light. Lane 1: molecular weight marker; lane 2: (FL-M23C)8, denatured in 7M GuHCl and labelled by IAEDANS; lane 3: (FL-M23C)8, reacted with IAEDANS in its native conformation; lane 4: (FL)8, reacted with IAEDANS (negative control); lane 5: (FL-C)8, reacted with IAEDANS in the native state (positive control). FL-C contains a C-terminal cysteine residue as well as a N-terminal cysteine residue, which are always exposed. (c) Photographs of (FL-M23C)8 hydrogels labelled with IAEDANS in PBS buffer under ultraviolet illumination. The inset is the fluorescence spectrum of the (FL-M23C)8 hydrogel labelled with IAEDANS. The dominant peak at 490 nm is characteristic of IAEDANS, while the shoulder peak at 410 nm originates from the dityrosine fluorescence. The characteristic greenish blue fluorescence of (FL-M23C)8 hydrogels strongly indicates that some FL-M23C domains are unfolded in the hydrogel. (d) Photographs of (FL-M23C)8 hydrogels labelled with IAEDANS in PBS buffer under white light. (e) Estimate of fraction of FL domains that are unfolded in the hydrogels by using the relative fluorescence intensity of IAEDANS-labelled hydrogels. In this measurement, the IAEDANS fluorescence intensity at 490 nm of dye-labelled, completely denatured FL-M23C hydrogels was taken as one, and the fluorescence intensity at 490 nm of unlabelled hydrogels was taken as zero. Assuming that the fluorescence intensity is linearly proportional to the concentration of IAEDANS-labelled FL, we calculated that ∼30% of FL domains are unfolded in the unstrained hydrogels. In addition, the fraction of unfolded FL domains increases as a function of strain. Error bars represent s.d. of the experimental data.
