Using delayed decoupling to attenuate residual signals in editing filters.

Kenneth A Marincin,Indrani Pal,Dominique P Frueh

doi:10.5194/mr-2-475-2021

Kenneth A Marincin, Indrani Pal + Show 1 more

Open Access

https://doi.org/10.5194/mr-2-475-2021

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Abstract

Isotope filtering methods are instrumental in biomolecular nuclear magnetic resonance (NMR) studies as they isolate signals of chemical moieties of interest within complex molecular assemblies. However, isotope filters suppress undesired signals of isotopically enriched molecules through scalar couplings, and variations in scalar couplings lead to imperfect suppressions, as occurs for aliphatic and aromatic moieties in proteins. Here, we show that signals that have escaped traditional filters can be attenuated with mitigated sensitivity losses for the desired signals of unlabeled moieties. The method uses a shared evolution between the detection and preceding preparation period to establish non-observable antiphase coherences and eliminates them through composite pulse decoupling. We demonstrate the method by isolating signals of an unlabeled post-translational modification tethered to an isotopically enriched protein.

Highlights

Nuclear magnetic resonance (NMR) has become a mainstay of biomolecular studies, notably because its non-invasive nature makes it suited to study interactions between biomolecules at the atomic level, such as protein–protein (Sprangers and Kay, 2007), protein–DNA/RNA (Kalodimos et al, 2004), and protein–small molecule interactions (Meyer and Peters, 2003)
nonribosomal peptide synthetases (NRPSs) employ domains called carrier proteins (CPs) to covalently tether substrates and shuttle them between partner catalytic domains. These domains are organized in contiguous modules, and substrates attached to CPs on sequential modules are condensed such that an upstream CP donates its substrate to a downstream CP, which harbors an extended intermediate
We focus on the 9.6 kDa carrier protein (PCP1) isolated from yersiniabactin synthetase, which natively harbors a cysteine substrate (Gehring et al, 1998)

Summary

Introduction

Nuclear magnetic resonance (NMR) has become a mainstay of biomolecular studies, notably because its non-invasive nature makes it suited to study interactions between biomolecules at the atomic level, such as protein–protein (Sprangers and Kay, 2007), protein–DNA/RNA (Kalodimos et al, 2004), and protein–small molecule interactions (Meyer and Peters, 2003). Isotopic filtering will enable the study of interactions between NRPSs and their substrates. NRPSs employ domains called carrier proteins (CPs) to covalently tether substrates and shuttle them between partner catalytic domains. These domains are organized in contiguous modules, and substrates attached to CPs on sequential modules are condensed such that an upstream CP donates its substrate to a downstream CP, which harbors an extended intermediate. We and others have found that some CPs interact transiently with their tethered substrates (Goodrich et al, 2015; Jaremko et al, 2015) such that the phosphopantetheine group and its attached sub-

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