Transmembrane segment enhanced labeling as a tool for the backbone assignment of α-helical membrane proteins

• Published in: Proceedings of the National Academy of Sciences - PNAS 2008-06, Vol.105 (24), p.8262-8267
• Main Authors: Reckel, Sina, Sobhanifar, Solmaz, Schneider, Birgit, Junge, Friederike, Schwarz, Daniel, Durst, Florian, Löhr, Frank, Güntert, Peter, Bernhard, Frank, Dötsch, Volker
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino acids; Biological Sciences; Carbon Isotopes - chemistry; Chemical equilibrium; Connectivity; Crystallography, X-Ray; Data collection; Detergents; Isotope Labeling - methods; Membrane proteins; Membrane Proteins - chemistry; Micelles; Molecular Sequence Data; Nitrogen Isotopes - chemistry; Nuclear Magnetic Resonance, Biomolecular - methods; Protein Structure, Secondary; Proteins; Spectroscopy; String theory
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0710843105

Recent advances in cell-free expression protocols have opened a new avenue toward high-resolution structural investigations of membrane proteins by x-ray crystallography and NMR spectroscopy. One of the biggest challenges for liquid-state NMR-based structural investigations of membrane proteins is the significant peak overlap in the spectra caused by large line widths and limited chemical shift dispersion of α-helical proteins. Contributing to the limited chemical shift dispersion is the fact that [almost equal to]60% of the amino acids in transmembrane regions consist of only six different amino acid types. This principle disadvantage, however, can be exploited to aid in the assignment of the backbone resonances of membrane proteins; by ¹⁵N/¹³C-double-labeling of these six amino acid types, sequential connectivities can be obtained for large stretches of the transmembrane segments where number and length of stretches consisting exclusively of these six amino acid types are enhanced compared with the remainder of the protein. We show by experiment as well as by statistical analysis that this labeling scheme provides a large number of sequential connectivities in transmembrane regions and thus constitutes a tool for the efficient assignment of membrane protein backbone resonances.