HOWTOs

Protocols

Labelling of Membrane Proteins

Selective labelling techniques reduce the severe resonance overlap observed for many MPs and simplify spectra. CECF offers full labelling flexibility with limited scrambling (some degree of scrambling has been observed in cases of aspartate, asparagine, glutamate, and glutamine). Apart from labelling single amino acids, sophisticated selective and combinatorial labelling schemes have been developed.

Transmembrane Segment (TMS) Enhanced Labelling for Backbone Assignment of Hydrophobic Regions

Figure 1: Spectral improvement by TMS-Labelling

Table 1:Chemical shift data for TMS labelled amino acids

This technique exploits the unique nature of the transmembrane α-helical amino acid composition by targeting primarily these for labelling (see Figure 1). Statistical studies have revealed that the amino acids alanine, leucine, valine, phenylalanine, isoleucine and glycine constitute nearly 60% of TMSs. Specific labelling of this amino acid subset will therefore simplify NMR spectra while predominantly providing information from residues located in the hydrophobic TMSs (see Figure 1). This allows the unambiguous assignment of unique stretches within these regions via typical 3D NMR measurements, such as HNCA and HNCACB, which detect the amino acid Cα and C;beta; resonances, respectively. Assignment can then be achieved according to the characteristic chemical shifts of these residues (see Table 1).

1. Produce a 20 amino acid mixture composed of the six ¹⁵N, ¹³C labelled TMS selected amino acids and the remaining 14 nonlabelled amino acids.

2. Use this amino acid mixture in the established cell-free expression system.

3. Obtain HNCA spectrum and follow with assignment

4. If required, obtain HNCACB spectrum to resolve ambiguities in assignment

Selective Combinatorial Labelling for Backbone Assignment of Sequential Amino acids Pairs

Figure 2: Example for a combinatorial labelling scheme

In this scheme, certain selected amino acids are ¹⁵N labelled and others ¹³C labelled. Three samples are then prepared, each having a different labelling combination (see Figure 2). Selective two-dimensional NMR experiments, namely HSQC and HNCO, are then measured. The HSQC experiment maps the position of the ¹⁵N amide resonance of each amino acid within the protein, whereas the HNCO experiment relies on the transfer of magnetism from the amide to the preceding carbonyl and back along the peptide bond in order to provide a signal. Therefore, one should observe all ¹⁵N amide resonances in the HSQC spectra, and only the resonances of those ¹⁵N amides which are preceded by a ¹³C carbonyl moiety in the HNCO spectra. These spectra together would then allow identification of amino acid pairs thus permitting specific assignments assuming that the pairs are unique.

1. Assign backbone as far as possible using standard NMR methods.

2. Choose regions where assignment is sparse and look for unique combinations of amino acid pairs. Group amino acids in three different samples in such a way that the chemical shift pattern in the six recorded spectra will be unique for each pair of amino acids.

3. Prepare a labelling scheme where the second amino acid in each pair is ¹⁵N labelled and the first, preceding amino acid is ¹³C labelled (see Figure 2).

4. Prepare 3 amino acid mixtures, where the selected amino acids are ¹⁵N and ¹³C labelled accordingly while all others remain unlabelled. The amino acid composition should reflect the percent composition of each amino acid in the given protein. Use this mix in the estrablished cell-free expression system.

5. Proceed with the measurement of 2D versions of the TROSYHSQC and HNCO experiments.