Rotational resonance NMR study of the active site structure in bacteriorhodopsin: con
 

Rotational resonance NMR study of the active site structure in bacteriorhodopsin: conformation of the Schiff base linkage.

Related Articles Rotational resonance NMR study of the active site structure in bacteriorhodopsin: conformation of the Schiff base linkage.

Biochemistry. 1992 Sep 1;31(34):7931-8

Authors: Thompson LK, McDermott AE, Raap J, van der Wielen CM, Lugtenburg J, Herzfeld J, Griffin RG

Rotational resonance, a new solid-state NMR technique for determining internuclear distances, is used to measure a distance in the active site of bacteriorhodopsin (bR) that changes in different states of the protein. The experiments are targeted to the active site of bR through 13C labeling of both the retinal chromophore and the Lys side chains of the protein. The time course of the rotor-driven magnetization exchange between a pair of 13C nuclei is then observed to determine the dipolar coupling and therefore the internuclear distance. Using this approach, we have measured the distance from [14-13C]retinal to [epsilon-13C]Lys216 in dark-adapted bR in order to examine the structure of the retinal-protein linkage and its role in coupling the isomerizations of retinal to unidirectional proton transfer. This distance depends on the configuration of the intervening C=N bond. The 3.0 +/- 0.2 A distance observed in bR555 demonstrates that the C=N bond is syn, and the 4.1 +/- 0.3 A distance observed in bR568 demonstrates that the C=N bond is anti. These direct distance determinations independently confirm the configurations previously deduced from solid-state NMR chemical shift and resonance Raman vibrational spectra. The spectral selectivity of rotational resonance allows these two distances to be measured independently in a sample containing both bR555 and bR568; the presence of both states and of 25% lipid in the sample demonstrates the use of rotational resonance to measure an active site distance in a membrane protein with an effective molecular mass of about 85 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)

PMID: 1510979 [PubMed - indexed for MEDLINE]



Source: PubMed