Bacteriorhodopsin assignment

Birge RR, Cooper TM, Lawrence AE, Masthay MB, Zhang CF, Zidovetzki R. Revised assignment of energy storage in the primary photochemical event in bacteriorhodopsin. J. Am. Chem. Soc. 1991 113 4327-4328. 

Fig. 1. Sequence alignment for three bacteriorhodopsins and two halorhodopsins. For brevity the single-letter amino acid code is used. Alignment and helical assignments are as in ref. [17]. Designations AR-1, a bacteriorhodopsin from Halobacterium sp. aus-1 [44] AR-2, a bacteriorhodopsin from Halobacterium sp. aus-2 [45] BR, bacteriorhodopsin from H. halobium [5] FIR, halorhodopsin from H. halobium [43] PHR, a halorhodopsin from Natronobacterium pharaonis[ l]. The patterns of dots and asterisks indicate either identity in all five sequences (dots only) or identity among the bacteriorhodopsins and halorhodopsins only (dots and asterisks).

TM) protein bacteriorhodopsin (bR) in oriented purple membranes. Our initial numerical studies indicated that the spectra from the two types of 2D experiments on uniformly N labeled bR would be too crowded to allow for assignment of all resonances.For this reason, our first experiments focused on samples with more diluted labeling patterns, specifically bR with N labeled methionines, i.e, Met-bR. Figure 13a shows the sequence and the tentative location of the secondary stmcture elements of bR highlighting the nine Met residues of which six or seven are expected to be in the helix region (Fig. 13b). The experimental PISEMA and HETCOR... 

One can dso obtain NMR spectra for proteins in micelles, which may allow the study of membrane protein structure in an environment approximating their native one. A combination of labels (l N,13c) was used for NMR studies of detergent-solubilized M13 coat protein. Although most of die resonances in the spectrum have not been assigned, there was clear indication that many of the protein residues had two distinct resonances of equal intensity. This was interpreted to mean (in combination with the results of sedimentation equilibrium, Raman and CD studies) that the protein was present in two conformers that represent the non-equivalent monomers of an asymmetric dimer. NMR has also been used to determine the spatial structures of gramicidin A and -labeled bacteriorhodopsin fragments in a membrane-like milieu b... 

A main difference in the modelling approach was the use of the structural data of bacteriorhodopsin, either employed only as a 3D template for building the transmembrane helices from helical sequences otherwise assigned [62-64, 66], or also taken as the basis for the identification of the helical sequences [65]. 

Ultrafast continuum pump-probe spectroscopy has led to the discovery of subpicosecond processes in bacteriorhodopsin. This protein contains a retinal chromophore and acts as a light-induced proton pump that creates a potential difference across a membrane in Halobacterium halobium, which can be used as a free-energy source. Changes in the bleaching, SE, and ESA were observed and were used in concert to describe, assign, and model the observed spectral changes and kinetics. 

Fig. 3. Resonance Raman spectrum of the all-trani-retinal protonated Schiff base chromo-phore in light-adapted bacteriorhodopsin. The assignments are of the various enhanced vibrational normal modes are indicated. (From Smith el al. )...

Varga K, Aslimovska L, Watts A (2008) Advances towards resonance assignments for uniformly - C-13, N-15 enriched bacteriorhodopsin at 18.8 T in purple membranes. J Biomol NMR 41 1 ... 

A general feature of dynamic aspect of membrane proteins has been revealed by site-directed NMR studies on bacteriorhodopsin as a typical membrane protein and a variety of mutants at ambient temperature. NMR signals in the spectrum of [3- C] Ala- or [1- C] Val-labelled proteins were assigned regio-specifically with reference to the data of the conformation-dependent chemi-... 

Table 3. Assigned C chemical shifts for [3- C]Ala and [l- C]Val residues in [3- C]Ala-, [l- C]Val-labeled bacteriorhodopsin from purple membrane...

Finally, in the past few years a large number of derivatives of natural retinaldehyde have been synthesized for use in the study of mechanistic details of the formation and function of rhodopsin (Vision Symposium, 1979 Sen et al., 1982 Liu and Asato, 1982) and bacteriorhodopsin (Ebrey and Yoshizawa, 1981). The efforts in the field of synthesis have been accompanied by the rapid development of analytical methods, especially NMR spectroscopy and high-performance liquid chromatography (HPLC), which permit rapid and reliable structure assignment. 

---