Membrane proteins pose a great challenge for structural biology. Structure determination of membrane proteins is difficult because many membrane proteins cannot be expressed in E. coli or other cellular systems in quantities necessary for structural investigations and because obtaining diffracting crystals or high resolution NMR spectra is problematic. Recently, we have shown that at least the expression problem can be solved for many membrane proteins by using cell-free expression systems. Based on these cell-free expression systems we have developed labelling methods with NMR active isotopes that reduce the peak overlap and enable us to obtain the backbone assignments of even larger membrane proteins. The first method, called TMS labelling (transmembrane segment enhanced labelling) is based on the observation that 60% of the amino acids found in the transmembrane helices belong to the following 6 amino acids: Ala, Leu, Ile, Phe, Gly and Val. TMS-labelled proteins contain these six amino acid types 15N/13C double labelled with all other amino acids 14N/12C labelled. This reduced labelling strategy significantly reduces the signal overlap, at the same time, however, allows for the assignment of large stretches of residues due to the clustering of these six amino acid types in the transmembrane regions. The figure shows the same plane taken from an HNCA of a uniformly double labelled sample (Fig.1, left) and a TMS-labelled sample (Fig.1, right). In the TMS-labelled sample connectivities can be identified which cannot be unambigously assigned due to severe overlap in the uniformly labelled sample.
A second labelling method that is based on cell-free expression of membrane proteins is combinatorial selective labelling. In this scheme a double labelling strategy in which one amino acid type is labelled with 15N and a second one with 13C is used. In 2D HNCO experiments peaks will only appear for amino acids combinations in which the 13C-lablled amino acid type is directly followed by the 15N-lablled type31-35. Provided that this combination exists only once in the entire protein sequence the single HNCO peak can be assigned to one specific amino acid. To obtain more than one assignment per sample we have developed a combinatorial labelling strategy that allows us to obtain more than one assignment per sample.
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- Koglin, A., Klammt, C., Trbovic, N., Schwarz, D., Schneider, B., Schäfer, B. Löhr, F., Bernhard, F. & Dötsch, V. Combination of cell-free expression and NMR spectroscopy as a new approach for structural investigation of membrane proteins. Magn. Reson. Chem., 44, 17-23 (2006)