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.

Hydrogen/deuterium exchange mass spectrometry and optical spectroscopy as complementary tools for studying the structure and dynamics of a membrane protein

“…The percentage of non-exchangeable amide hydrogens in the bleached membranes is 66.6%. Our findings are consistent with previous 13 C-NMR [47,80], AFM [48], FRET [81] and infrared studies [22], all of which indicate that the protein structure in the bleached membrane remains similar to that of native BR, despite the loss of membrane crystallinity and the occurrence of greater disorder in some local regions. Thus, major aspects of the native BR structure are not dependent on the presence of an intact covalent linkage between protein and chromophore.…”

“…but that structural perturbations persist for both the retinal-bound (89%) and the retinal-free (11%) forms. NMR data suggest that some of this disorder is attributable to loop elements that do not fully recover during BR folding in vitro [80,82]. Our findings suggest that the chromophore plays a relatively minor role in guiding the protein along its folding pathway, at least as far as the formation of secondary structure is concerned [83].…”

Characterization of photo-intermediates in the photo-reaction pathways of a bacteriorhodopsin Y185F mutant using in situ photo-irradiation solid-state NMR spectroscopy

“…Isotopically labeled regenerated Y185F-bRs were obtained by the addition of [20-13 C, 14- 13 C] retinal or [20-13 C, 15-13 C]retinal to Y185F-bOs obtained from bleached Y185F-bR in a process termed in vitro retinal reconstitution. 29 Retinal bleaching and reconstitution were confirmed by measuring the absorbance of bR. Samples were then suspended in a 5 mM N-(2-hydroxyethyl)piperazine-N′-2-ethane sulfonic acid (HEPES) buffer and 10 mM NaCl at pH 7.…”

Photoreaction Pathways of Bacteriorhodopsin and Its D96N Mutant as Revealed by in Situ Photoirradiation Solid-State NMR

“…D96N bacterio-opsins (D96N-BOs) were prepared by photo-bleaching of the corresponding D96N-BR in hydroxylamine solution (500 mM, pH 7) at 4 °C under white light illumination for one day. Isotopically labeled regenerated [20- 13 C, 14- 13 C]retinal-D96N-BRs were obtained by the addition of [20- 13 C, 14- 13 C]retinal to the D96N-BOs obtained from bleached D96N-BR in a process termed in vitro retinal reconstitution [ 47 ]. Retinal bleaching and reconstitution were confirmed by measuring the absorbance of D96N-BR.…”