title: Participation of the BC loop in the correct folding of bacteriorhodopsin as revealed by solid-state NMR.
abstract: Structural changes in bacteriorhodopsin (bR) in two different processes of retinal reconstitutions were investigated by observing the (13)C and (15)N solid-state NMR spectra of [1-(13)C]Val- and [(15)N]Pro-labeled bR. We found that NMR signals of the BC loop were sensitive to changes in protein structure and dynamics, from wild-type (WT) bR to bacterio-opsin (bO), regenerated bR and E1001 bR. Regenerated bR was prepared following the addition of retinal into bO obtained from photobleached WT-bR. E1001 bR was cultured from a retinal-deficient strain termed E1001 following the addition of retinal to growing cells. (15)N NMR signal at Pro70 in the BC loop in WT-bR was observed at 122.4 p.p.m., whereas signals were not apparent or partly suppressed in bO and regenerated bR, respectively. Similarly, the (13)C NMR signal at Val69 in the BC loop at 172.0 p.p.m. that was observed in WT-bR was significantly decreased in both regenerated bR and bO. These results suggest that the dynamic structure of the BC loop in bO was substantially altered following the removal of retinal. As a consequence, the correct protein structure failed to be recovered via the regenerating process of retinal to bO. On the other hand, (13)C and (15)N NMR signals at the BC loop in E1001 bR appeared at positions identical to those of WT-bR. The results of the current study indicate that the BC loop may not always fold correctly in the regenerated bR, which leads to different properties in the regenerated bR compared to that of WT-bR.
authors.affiliation: Graduate School of Engineering, Yokohama National University, Hodogaya-ku, Yokohama, Japan.
authors.forename: Izuru
authors.initials: I
authors.lastname: Kawamura
authors.forename: Junko
authors.initials: J
authors.lastname: Tanabe
authors.forename: Masato
authors.initials: M
authors.lastname: Ohmine
authors.forename: Satoru
authors.initials: S
authors.lastname: Yamaguchi
authors.forename: Satoru
authors.initials: S
authors.lastname: Tuzi
authors.forename: Akira
authors.initials: A
authors.lastname: Naito
mesh_terms: D000595:Amino Acid Sequence; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019617:Halobacterium salinarum / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D017510:Protein Folding; D017434:Protein Structure, Tertiary
chemical_list: D001436:Bacteriorhodopsins