The Conformation of Bacteriorhodopsin Loops in Purple Membranes Resolved by Solid‐State MAS NMR Spectroscopy

Abstract

Making complements: Solid‐state MAS NMR spectra of bacteriorhodopsin in its native purple membrane environment can be used to complement crystallographic studies of the protein by validating and redefining the (possibly distorted) loop structures. Backbone dihedral angles were extracted from the chemical shifts and compared to the crystal structures. Where there are conformational differences, the dihedral angles were used to recalculate the loop structure (see picture).

Novel expression and characterization of a light driven proton pump archaerhodopsin 4 in a Halobacterium salinarum strain

“…6C is a 13 C-15 N DCP correlation experiment on [ 13 C 9 , 15 N]-Leu labeled native AR4 (black) and recombinant AR4 (red). The chemical shifts of the backbone 13 CO, 13 CA and 15 N for the recombinant AR4 are all in the ranges typical for a well formed α-helical structure, and in a good agreement with chemical shifts of Leu residues in BR and other retinal proteins by both solution NMR and solid-state NMR [63][64][65][66]. Spinning sidebands from the CO peaks in all the 13 C-13 C correlation experiments of AR4 clearly indicate that AR4 has a relative rigid transmembrane domain than that of recombinant AR4 which may attribute to the effect of bacterioruberin on the native AR4, as shown by the projections for both t 1 and t 2 dimensions to the 2D spectra in Fig.…”

“…The 2D DQ/SQ 13 C-13 C correlation spectra of the 13 C leu labeled native and recombinant AR4 also show slightly up-field shifted CO and CA peaks, which may indicate the proteins may have a similar structural feature. Our results further demonstrate that this expression method can provide a platform for the structure determination and functional analysis of AR4 by solid-state NMR [66,[69][70][71][72][73][74][75][76].…”

Helical membrane protein conformations and their environment

“…Once again it is important to recognize that despite the imperfections in the crystal environments, XRD has resulted in many structures that appear native-like from the perspective of the TM domain. We know much less at this time about the structure and dynamics of membrane proteins in the membrane interfacial region (Higman et al 2011), a topic that is beyond the scope of this review. For the TM domain it appears that when crystal lattices take on a bilayer appearance (Schulz 2011) and when lipids are integrated into the membrane protein environment the probability for a native-like structure increases.…”

“…There are two techniques for achieving membrane protein structural restraints from solid state NMR (ssNMR); Magic Angle sample Spinning (MAS) (Huang and McDermott 2008; Andreas et al 2010; Li et al 2008; Lange et al 2006; Higman et al 2011) and oriented sample (OS) spectroscopy (Watts et al 2004; Ketchem et al 1993; Cross and Opella 1994; Fu and Cross 1999). Structural restraints have been obtained for numerous membrane proteins using these techniques, with most success being reported for small TM proteins, and assignments reported for selected regions of large proteins in bilayers.…”

Recent Solid-State NMR Studies of Membrane-Bound Peptides and Proteins

“…The structures of the BC and AB loops were further determined using uniformly [ 13 C, 15 N]-labeled bR by means of solid-state MAS NMR spectroscopy [162]. The residues of all of the loops could be detected in CP-based spectra, indicating that the loops in bR are relatively rigid, finding that was in agreement with the relaxation measurements [161].…”