NMR experiments on the transient interaction of the intrinsically disordered N-terminal peptide of cystathionine-β-synthase with heme

Abstract

The N-terminal segment of human cystathionine-β-synthase (CBS(1-40)) constitutes an intrinsically disordered protein stretch that transiently interacts with heme. We illustrate that the HCBCACON experimental protocol provides an efficient alternative approach for probing transient interactions of intrinsically disordered proteins with heme in situations where the applicability of the conventional [¹H, ¹⁵N]-HSQC experiment may be limited. This experiment starting with the excitation of protein side chain protons delivers information about the proline residues and thereby makes it possible to use these residues in interaction mapping experiments. Employing this approach in conjunction with site-specific mutation we show that transient heme binding is mediated by the Cys¹⁵-Pro¹⁶ motif of CBS(1-40).

Keywords: Cystinuria; HCBCACON experiment; Heme; Heteronuclear NMR spectroscopy; Human cystathionine-β-synthase; Mapping; Transient binding.