Abstract
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.
Highlights
Forming a multitude of temporary H-bonds, H2O molecules interact with backbone amides and loosen the polar intramolecular interactions holding together proteins’ secondary and tertiary structures
The temperature dependent Far UV range (FUV)-Electronic circular dichroism spectroscopy (ECD) spectra for all 4 SS-bond enforced model peptides were recorded between 5 °C and 85 °C (SFigure 1)
E11 was found to be more helical than the longer E19 [56], we find here that both E11_SS and E11_2SH have more compact α-helices than E19_SS and E19_2SH, according to both the ΣCSD(Hi)α Nuclear magnetic resonance spectroscopy (NMR)-measure and the FUV ECD-spectral i properties
Summary
Forming a multitude of temporary H-bonds, H2O molecules interact with backbone amides and loosen the polar intramolecular interactions (salt-bridges, H-bonds, cation/anion↔π-interactions, etc.) holding together proteins’ secondary and tertiary structures. The effect of the compactness on the protein fold, the accessibility and the local explicit charges of the SS-bond, the reagent type on reduction rate and the mechanism are explanted in this paper.
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