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

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 <i>in vitro</i> synthesis of various subunits, each with a different isotopic labelling scheme. We exploited the open nature of the cell-free synthesis expression system by introducing a stable subunit directly into the synthesis of the unstable or poorly soluble component of the complex. This protocol was used to produce the ClpXP 26-subunit complex, in which the methyl groups were labelled on either ClpP or ClpX. The stabilisation of the newly synthesised ClpX subunits by the ClpP core enabled production of a stable and functional 760 kDa AAA+ proteolytic machine. ¹³CH₃-labelling of the alanine and methionine in ClpXP allowed acquisition of high quality 2D solution NMR spectra, the characterization of the oligomeric size using diffusion-ordered NMR spectroscopy, and observation of structural rearrangements induced by nucleotide binding.