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Compactness of Protein Folds Alters Disulfide‐Bond Reducibility by Three Orders of Magnitude: A Comprehensive Kinetic Case Study on the Reduction of Differently Sized Tryptophan Cage Model Proteins
Author(s) -
Horváth Dániel,
Taricska Nóra,
Keszei Ernő,
Stráner Pál,
Farkas Viktor,
Tóth Gábor K.,
Perczel András
Publication year - 2020
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201900470
Subject(s) - steric effects , chemistry , circular dichroism , disulfide bond , peptide bond , crystallography , peptide , stereochemistry , biochemistry
A new approach to monitor disulfide‐bond reduction in the vicinity of aromatic cluster(s) has been derived by using the near‐UV range ( λ =266–293 nm) of electronic circular dichroism (ECD) spectra. By combining the results from NMR and ECD spectroscopy, the 3D fold characteristics and associated reduction rate constants ( k ) of E19_SS, which is a highly thermostable, disulfide‐bond reinforced 39‐amino acid long exenatide mimetic, and its N‐terminally truncated derivatives have been determined under different experimental conditions. Single disulfide bond reduction of the E19_SS model (with an 18‐fold excess of tris(2‐carboxyethyl)phosphine, pH 7, 37 °C) takes hours, which is 20–30 times longer than that expected, and thus, would not reach completion by applying commonly used reduction protocols. It is found that structural, steric, and electrostatic factors influence the reduction rate, resulting in orders of magnitude differences in reduction half‐lives (900> t 1/2 >1 min) even for structurally similar, well‐folded derivatives of a small model protein.