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  • Title: Solid-state 13C-NMR of [(3-13C)Pro]bacteriorhodopsin and [(4-13C)Pro]bacteriorhodopsin: evidence for a flexible segment of the C-terminal tail.
    Author: Engelhard M, Finkler S, Metz G, Siebert F.
    Journal: Eur J Biochem; 1996 Feb 01; 235(3):526-33. PubMed ID: 8654397.
    Abstract:
    The configuration of an Xaa-Pro bond can be determined by solid-state magic-angle-sample-spinning (MASS)-13C-NMR spectroscopy since the chemical shifts of C beta and Cgamma of the proline ring are sensitive to the isomerization state of the preceding peptide bond. (3-13C)Pro and (4-13C)Pro have been chemically synthesized; the former by means of an asymmetric synthesis. The 13C-labeled Pro residues were biosynthetically incorporated into bacteriorhodopsin with a yield of 80%. The solid-state-MASS-13C-NMR spectra of [(3-13C)Pro]bacteriorhodopsin and [(4-13C)Pro]bacteriorhodopsin revealed isotropic chemical shifts at 29.8 ppm and 25.5 ppm, respectively. From the chemical-shift values we conclude that all Xaa Pro peptide bonds are in the trans configuration confirming previous results from solution-NMR studies on solubilized bacteriorhodopsin in organic solvents [Deber, M.C., Sorrell, B.J. & Xu, G.Y. (1990) Biochem. Biophys. Res. Commun. 172, 862-869]. Inversion-recovery experiments could differentiate between three classes of Pro residues distinguished by their relaxation time t1. Tentatively, these three distinct groups of Pro residues could be assigned to the helical, the loop, and the C-terminal parts of the protein. The resonances of the two C-terminal Pro could be identified by removing the C-terminus by proteolysis. Although they are separated by only one Glu they occupy different chemical environments and possess different flexibilities. These results indicate that the first part of the C-terminal tail is constrained. Pro238 marks the position where the tail becomes freely mobile. It is proposed that the C-terminus is fixed to the membrane via salt bridges between divalent cations and negative charges of the C-terminus as well as interhelical loops.
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