Backbone Structures and Dynamics in Monomers

It is expected, however, that the backbone dynamics could be substantially modified when such a 2D lattice assembly is distorted or disrupted as in bacterio-opsin (bO) prepared from either an hydroxylamine-treated bR or retinal-deficient ElOOl strain in which retinal-helix interactions is absent owing to lack of retinal. The resultant protein dynamic change caused by the removed retinal through the modified helix-helix interaction resulted in the preferentially suppressed NMR signals of [3-Ala-labeled bO at the loops and transmembrane a-helices near to the membrane surface. At the same time, the NMR spectrum of [l- C]Val-labeled bO was also partly suppressed especially in the region of the loops at lower frequency by acquisition of the fluctuation motions. 

In addition, the C NMR signals from the loops and several C NMR peaks from the transmembrane a-helices near to the surface, including Ala 39, 53, 168, and 215 were preferentially suppressed in these mutants as encountered when retinal-protein interactions are substantially modified as in the case of D85N mutant or they are absent as in bO. This is due to the acquired internal fluctuation with a correlation time of 10 s, which interferes with the proton decoupling frequency of 50 kHz. Further, it should be noted that the C CP-MAS 

It is anticipated that oligomerization as well as lattice formation is not always straightforward for a number of membrane proteins overexpressed in a host cell like E. coli, unless identiflcation and incorporation of such specific endogenous lipids to promote the formation of a 2D lattice are seriously taken into account. As a reference system to examine the NMR spectra of such proteins by site-directed NMR approach, it seems to be very important to gain an insight into how the NMR spectra of [3- C]Ala- or [l- C]Val-labeled bR incorporated into neutral lipid bilayers such as egg phosphatidylcholine (PQ, dimyristoylphosphatidylcholine (DMPC) or dipalmytoylphosphatidylcholine (DPPC) are modified by the presence or absence of a 2D crystalline lattice. 

In the presence of such endogeneous lipids, it is anticipated that the 2D lattiee assembly could be formed when the temperature of bR in neutral lipid bilayers is lowered to that of the gel phase in the presence of higher ionic strengths. Saito et showed that this is the case at low temperature in the presence of a 10 mM 

In the case of [l- C]Val-bR, which is sensitive to fluctuation motions of 10 Hz, this condition is more stringent than that of [3- C]Ala-bR, which is sensitive to fluctuation motions of 10 Hz. A NMR spectral pattern characteristic of 2D lattice is achieved at 0°C as viewed from the [l- C]Val-labeled bR in the DPPC bilayer (Fig. 30C), while this feature is achieved from the [3- C]Ala-labeled bR at 20°C (Fig. 30B). Therefore, a suitable choice of C-labeled amino acid, either [3- C]Ala or [l- C]Val as well as the manner of protein aggregation is very important for the study of the conformation and dynamics of membrane proteins by the site-directed NMR approach, as summarized in Tables 7 and 8. 

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