Fig. 3.

Conformational properties of the NTD in αB-3E. (A) Quenching of intrinsic tryptophan fluorescence by acrylamide. Fluorescence of the intrinsic Trp probes (Trp9, Trp60) of αB-WT (black squares) and αB-3E (red circles) was quenched by acrylamide. The Stern-Volmer plot (relative decrease in fluorescence [F₀/F] vs. quencher concentration) shows more quenching for the tryptophan residues in the context of αB-3E ensemble indicating higher accessibility. (B) Overall surface hydrophobicity of αB-WT (black) and αB-3E (red) probed by ANS fluorescence. Note the increased fluorescence intensity of αB-3E compared with αB-WT indicating a gain in overall surface hydrophobicity. (Inset) Hydrophobicity plot of αB sequence. The sequence segment representing the NTD is colored orange. The hydrophobicity score was calculated on the basis of a hydrophobicity scale by Eisenberg et al. (70) using the web-based application ProtScale (71). (C) Limited proteolysis of αB-WT (Left) and αB-3E (Right) by α-chymotrypsin. Protein samples (10 µM) were incubated with α-chymotrypsin at a 1:25 (wt/wt) ratio. Proteolysis reactions were terminated after various time points and analyzed by SDS/PAGE. The cleavage products were identified using LC-MS. The respective bands are indicated by an arrow and labeled with the identified peptide. Samples without chymotrypsin (Neg) were included as control. LMW, low-molecular-weight marker.