Subunit-specific isotope labelling of heteromeric complexes using cell-free protein expression: application to the 760 kDa ClpXP molecular machine.

ABSTRACT: The specific insertion of methyl (¹³CH₃) probes into deuterated proteins enables study of the structure, dynamics, and mechanisms of symmetric complexes up to 1 MDa by solution nuclear magnetic resonance (NMR). For asymmetric or heteromeric high molecular weight complexes, subunit-specific labelling is required to simplify spectra, reduce resonance overlap, and facilitate data interpretation. However, the instability of one component may prevent the reconstitution of a functional complex with a single labelled subunit. Here, we employed a simple, multi-step expression protocol that enabled the sequential in vitro synthesis of various subunits, each with a different isotopic labelling scheme. We exploited the open nature of the cell-free synthesis expression sys

SUBMITTER: Audibert A 

PROVIDER: S-EPMC12683673 | biostudies-literature | 2025 Dec

REPOSITORIES: biostudies-literature