A simple protocol for controlling protein deuteration for neutron scattering.

Protein deuteration is a crucial technique in biological studies using neutron scattering/diffraction. In neutron crystallography, the use of deuterated proteins not only reduces the background due to hydrogen atoms but also prevents the cancellation of the scattering length density due to the negative density of hydrogen atoms. In small-angle neutron scattering, it allows the structures of individual components of a protein complex to be studied. Furthermore, in quasielastic neutron scattering, it allows one to measure the dynamics of specific components (or regions) in protein complexes (or a protein). Protocols for protein deuteration employ the expression system in E. coli cultured either in minimal media containing, for example, deuterated glycerol or in rich media containing deuterated algal hydrolysate (algal peptone). While the protocols using the minimal media are the mainstream, the protocols using the rich media allow easy control of deuteration. Here, we optimize the procedure for preparing algal peptone, and describe a protocol for protein deuteration using algal peptone. It is shown that ∼96 % deuteration of the test protein, α-synuclein (αSyn), can be routinely achieved using this protocol. The similarity of the structures of deuterated and hydrogenated αSyn was verified by the small-angle X-ray scattering measurements. Furthermore, the degrees of deuteration can be controlled simply by mixing the hydrogenated and deuterated peptone in the media, and hydrogen labeling of specific amino-acid residues is possible by adding hydrogenated amino acids to the media. The protocol described here is thus useful as a complementary method to those using the minimal media.