Abstract
We recently introduced RAP (reduced adjoining protonation) labelling as an easy to implement and cost-effective strategy to yield selectively methyl protonated protein samples. We show here that even though the amount of H2O employed in the bacterial growth medium is rather low, the intensities obtained in MAS solid-state NMR 1H,13C correlation spectra are comparable to spectra obtained for samples in which α-ketoisovalerate was employed as precursor. In addition to correlations for Leu and Val residues, RAP labelled samples yield also resonances for all methyl containing side chains. The labelling scheme has been employed to quantify order parameters, together with the respective asymmetry parameters. We obtain a very good correlation between the order parameters measured using a GlcRAP (glucose carbon source) and a α-ketoisovalerate labelled sample. The labelling scheme holds the potential to be very useful for the collection of long-range distance restraints among side chain atoms. Experiments are demonstrated using RAP and α-ketoisovalerate labelled samples of the α-spectrin SH3 domain, and are applied to fibrils formed from the Alzheimer’s disease Aβ1-40 peptide.
Highlights
Methyl groups are valuable probes for structure and dynamics investigations of proteins and possess favorable relaxation properties, such as short T1 and long T2 times in deuterated microcrystalline protein samples
One major benefit of the 5% GlcRAP labelling is that all methyl groups (Ala, Ile, Leu, Met, Thr, Val) become observable at once with comparable resolution, while in the Leu/Val labelling only two out of six methyl-bearing amino acids can be detected (Fig. 1D)
Since the 5% GlcRAP sample was uniformly 13C labelled, evolution of 13C,13C scalar couplings affects the resolution in the methyl region, except for methionine methyl groups, which lack an adjacent carbon (Fig. 1A)
Summary
Methyl groups are valuable probes for structure and dynamics investigations of proteins and possess favorable relaxation properties, such as short T1 and long T2 times in deuterated microcrystalline protein samples. We have subsequently observed that high resolution 1H,13C correlation spectra can be acquired in case the glucose employed in the bacterial growth medium is only 97% deuterated[11] This triggered the development of RAP (reduced adjoining protonation) labelling schemes in which H2O was added to the deuterated minimal medium which contained 2H,13C glucose[12]. The RAP labelled sample, yields labelling of all methyl groups with similar enrichment of protons for all methyl bearing side chains We believe that this labelling scheme will be employed widely in the future as it is easy to implement, allows to obtain assignments via HCCH type experiments[18], as well as information on local dynamics and structure
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