Open AccessSolid‐state 13 C‐NMR of [(3‐ 13 C)Pro]bacteriorhodopsin and [(4‐ 13 C)Pro]bacteriorhodopsin
Author(s) -
Engelhard Martin,
Finkler Stefan,
Metz Günther,
Siebert Friedrich
Publication year - 1996
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1996.00526.x
Subject(s) - bacteriorhodopsin , chemistry , chemical shift , crystallography , nuclear magnetic resonance spectroscopy , carbon 13 nmr , stereochemistry , solid state nuclear magnetic resonance , isomerization , magic angle spinning , nuclear magnetic resonance , organic chemistry , catalysis , membrane , physics , biochemistry
The configuration of an Xaa‐Pro bond can be determined by solid‐state magic‐angle‐sample‐spinning (MASS)‐ 13 C‐NMR spectroscopy since the chemical shifts of C β and C γ of the proline ring are sensitive to the isomerization state of the preceding peptide bond. (3‐ 13 C)Pro and (4‐ 13 C)Pro have been chemically synthesized; the former by means of an asymmetric synthesis. The 13 C‐labeled Pro residues were biosynthetically incorporated into bacteriorhodopsin with a yield of 80%. The solid‐state‐MASS‐ 13 C‐NMR spectra of [(3– 13 C)Pro]bacteriorhodopsin and [(4‐ 13 C)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 t 1 . 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.