Proteins’ fold compactness alters disulfide-bond reducibility by 3 orders of magnitude: a comprehensive kinetic case study on the reduction of different size Trp-cage model proteins

Horváth, Dániel and Taricska, Nóra and Keszei, Ernő and Stráner, Pál and Farkas, Viktor and Tóth, Gábor K. and Perczel, András (2019) Proteins’ fold compactness alters disulfide-bond reducibility by 3 orders of magnitude: a comprehensive kinetic case study on the reduction of different size Trp-cage model proteins. CHEMBIOCHEM. ISSN 1439-4227 (In Press)

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Abstract

We derived a novel approach to monitor disulfide bond reduction in the vicinity of aromatic cluster(s) by using the near-UV range (266–293 nm) of ECD spectra. By using combined NMR- and ECD-spectroscopy we have determined the 3D-fold characteristics and the associated reduction rate constants (k) of E19_SS – a highly thermostable; disulfide bond reinforced 39-amino acid long Exenatide mimetic – and its N-terminally truncated derivatives at different experimental conditions. Single SS-bond reduction of the E19_SS model (using 18-fold excess of TCEP, pH = 7, 37 °C) takes hours, 20-30 times longer than expected, thus would not reach completion applying the commonly used reduction protocols. We found that structural, steric and electrostatic factors influence the reduction rate, resulting in magnitude differences in reduction halftimes (900 > t½ > 1 min) even for structurally similar, well-folded derivatives of a small model-protein.

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