Conformation and backbone dynamics of bacteriorhodopsin revealed by (13)C-NMR.

It is demonstrated here how the secondary structure and dynamics of transmembrane helices, as well as surface residues, such as interhelical loops and N- or C-terminus of bacteriorhodopsin (bR) in purple membrane, can be determined at ambient temperature based on very simple (13)C-NMR measurements, together with a brief experimental background. In contrast to the static picture of bR, currently available from X-ray diffraction or cryo-electron microscopy, the structure consists of dynamically heterogeneous domains which undergo various types of local fluctuations with a frequency range of 10(2)--10 (8) Hz. The significance of this picture is discussed in relation to the biological function of this protein.

Evidence of local conformational fluctuations and changes in bacteriorhodopsin, dependent on lipids, detergents and trimeric structure, as studied by 13C NMR.

We examined how the local conformation and dynamics of [3-13C]Ala-labeled bacteriorhodopsin (bR) are altered as viewed from 13C NMR spectra when the natural membrane lipids are partly or completely replaced with detergents. It turned out that the major conformational features of bR, the alphaII-helices, are generally unchanged in the delipidated or solubilized preparations. Upon partial delipidation or detergent solubilization, however, a significant conformational change occurs, ascribed to local conversion of alphaII-->alphaI-helix (one Ala residue involved), evident from the upfield displacement of the transmembrane helical peak from 16.4 ppm to 14.5 ppm, conformational change (one or two Ala residues) within alphaII-helices from 16.4 to 16.0 ppm, and acquired flexibility in the loop region (especially at the F-G loop) as manifested from suppressed peak-intensities in cross-polarization magic angle spinning (CP-MAS) NMR spectra. On the other hand, formation of monomers as solubilized by Triton X-100, Triton N-101 and n-dodecylmaltoside is characterized by the presence of a peak at 15.5 ppm and a shifted absorption maximum (550 nm). The size of micelles under the first two conditions was small enough to yield 13C NMR signals observable by a solution NMR spectrometer, although 13C CP-MAS NMR signals were also visible from a fraction of large-sized micelles. We found that the 16.9 ppm peak (three Ala residues involved), visible by CP-MAS NMR, was displaced upfield when Schiff base was removed by solubilization with sodium dodecyl sulfate, consistent with our previous finding of bleaching to yield bacterioopsin.

Irreversible conformational change of bacterio-opsin induced by binding of retinal during its reconstitution to bacteriorhodopsin, as studied by (13)C NMR.

We compared (13)C NMR spectra of [3-(13)C]Ala- and [1-(13)C]Val-labeled bacterio-opsin (bO), produced either by bleaching bR with hydroxylamine or from a retinal-deficient strain, with those of bacteriorhodopsin (bR), in order to gain insight into the conformational changes of the protein backbone that lead to correct folding after retinal is added to bO. The observed (13)C NMR spectrum of bO produced by bleaching is not greatly different from that of bR, except for the presence of suppressed or decreased peak-intensities. From careful evaluation of the intensity differences between cross polarization magic angle spinning (CP-MAS) and dipolar decoupled-magic angle spinning (DD-MAS) spectra, it appears that the reduced peak-intensities arise from reduced efficiency of cross polarization or interference of internal motions with proton decoupling frequencies. In particular, the E-F and F-G loops and some transmembrane helices of the bleached bO have acquired internal motions whose frequencies interfere with proton decoupling frequencies. In contrast, the protein backbone of the bO from the retinal-negative cells is incompletely folded. Although it contains mainly a-helices, its very broad (13)C NMR signals indicate that its tertiary structure is different from bR. Importantly, this changed structure is identical in form to that of bleached bO from wild-type bR after it was regenerated with retinal in vitro, and bleached with hydroxylamine. We conclude that the binding of retinal is essential for the correct folding of bR after it is inserted in vitro into the lipid bilayer, and the final folded state does not revert to the partially folded form upon removal of the retinal.

Stability of the C-terminal alpha-helical domain of bacteriorhodopsin that protrudes from the membrane surface, as studied by high-resolution solid-state 13C NMR.

We have recorded 13C NMR spectra of [1-(13)C]Ala- and [3-(13)C]Ala-bacteriorhodopsin (bR), [1-(13)C]Ala- and [3-(13)C]Ala-papain-cleaved bR, and [3-(13)C]Ala-labeled R227Q bR mutant by cross polarization-magic angle spinning (CP-MAS) and dipolar decoupled-magic angle spinning (DD-MAS) methods. The pH and temperature were varied, and Arg 227 was replaced with Gln (R227Q), in order to clarify their effects on the stability of the alpha-helical domain of the C-terminus that protrudes from the membrane surface. The comparative 13C CP- and DD-MAS NMR study of [3-(13)C]Ala-bR, rather than [1-(13)C]Ala-bR, turned out to be the best means to distinguish the 13C NMR signals of the C-terminus from those of the rest of the transmembrane helices or loops. The inner segment of the C-terminus, from Ala 228 to Ala 235, forms an alpha-helical domain (resonated at 15.9 ppm) either at neutral pH and/or at 10 to -10 degrees C. The alpha-helical peak was not seen, however, after either cleavage of the C-terminus with papain or lowering the pH to 4.25. This alpha-helical structure, and a part of the random coil which was produced from the helix at pH 4.25, were further converted to a low-temperature-type alpha-helix, as indicated by an upfield displacement of the 13C NMR signal, when the temperature was lowered to 10- -10 degrees C. Surprisingly, the corresponding helical structure in R227Q is more stable than in the wild type at the acidic pH. This alpha-helical peak was classified as an alphaII-helix from the 13C chemical shifts of Cbeta carbon, although it was ascribed to an alphaI-helix on the basis of the carbonyl shifts. This is in contrast to Ala 53 which adopts the alphaII-helix as judged from the 13C chemical shifts of Cbeta and the carbonyl carbons. Therefore, this discrepancy might be caused by differential sensitivity of the two types of carbon signals to conformation and to modes of hydrogen bonding when motional fluctuation is involved. It is likely that the alphaII-helix form present at the C-terminus is not always the type originally proposed but should be considered as a form undergoing large-amplitude conformational fluctuation around alpha-helix.

A method for provoking EEG abnormalities by administration of nialamide.

Nialamide, a kind of monoamine oxydase inhibitor, was used for provoking centrencephalic EEG abnormalities and 6-14 Hz positive spikes. It was assumed that these EEG abnormalities have close relationship with dysfunctions of the brainstem which is rich in monoamines and monoamine oxydase. The nialamide provocation was carried out on 49 inpatients who had centrencephalic discharges in their EEG reports. These patients consisted of four cases of epilepsy, one case of anorexia nervosa, two cases of narcolepsy and 42 cases of diencephalosis. Another series of 22 patients suffering from other diseases, in whome EEG no centrencephalic EEG abnormallities were detected, were examined with nialamide. Out of the 49 patients, 35 cases (71.4+) showed increased EEG abnormalities following the administration of nialamide. It was noteworthy that this drug had provocative effect not only for EEG abnormalities, but also for the symptoms from which patients were suffering. In 26 out of 49 patients, both EEG abnormalities and clinical symptoms were provoked by nialamide. And the nialamide administration resulted in negative on all 22 patients who did not register centrencephalic EEG abnormalities. The mechanism and characteristics of this provocative procedure by the use of nialamide were evaluated.

Conformational changes of bacteriorhodopsin along the proton-conduction chain as studied with (13)C NMR of [3-(13)C]Ala-labeled protein: arg(82) may function as an information mediator.

We have recorded (13)C NMR spectra of [3-(13)C]Ala-labeled wild-type bacteriorhodopsin (bR) and its mutants at Arg(82), Asp(85), Glu(194), and Glu(204) along the extracellular proton transfer chain. The upfield and downfield displacements of the single carbon signals of Ala(196) (in the F-G loop) and Ala(126) (at the extracellular end of helix D), respectively, revealed conformational differences in E194D, E194Q, and E204Q from the wild type. The same kind of conformational change at Ala(126) was noted also in the Y83F mutant, which lacks the van der Waals contact between Tyr(83) and Ala(126) present in the wild type. The absence of a negative charge at Asp(85) in the site-directed mutant D85N induced global conformational changes, as manifested in displacements or suppression of peaks from the transmembrane helices, cytoplasmic loops, etc., as well as the local changes at Ala(126) and Ala(196) seen in the other mutants. Unexpectedly, no conformational change at Ala(126) was observed in R82Q (even though Asp(85) is protonated at pH 6) or in D85N/R82Q. The changes induced in the Ala(126) signal when Asp(85) is uncharged could be interpreted therefore in terms of displacement of the positive charge of Arg(82) toward Tyr(83), where Ala(126) is located. It is possible that disruption of the proton transfer chain after protonation of Asp(85) in the photocycle could cause the same kind of conformational change we detect at Ala(196) and Ala(126). If so, the latter change would be also the result of rearrangement of the side chain of Arg(82).

Alteration of conformation and dynamics of bacteriorhodopsin induced by protonation of Asp 85 and deprotonation of Schiff base as studied by 13C NMR.

According to previous X-ray diffraction studies, the D85N mutant of bacteriorhodopsin (bR) with unprotonated Schiff base assumes a protein conformation similar to that in the M photointermediate. We recorded (13)C NMR spectra of [3-(13)C]Ala- and [1-(13)C]Val-labeled D85N and D85N/D96N mutants at ambient temperature to examine how conformation and dynamics of the protein backbone are altered when the Schiff base is protonated (at pH 7) and unprotonated (at pH 10). Most notably, we found that the peak intensities of three to four [3-(13)C]Ala-labeled residues from the transmembrane alpha-helices, including Ala 39, 51, and 53 (helix B) and 215 (helix G), were suppressed in D85N and D85N/D96N both from CP-MAS (cross polarization-magic angle spinning) and DD-MAS (dipolar decoupled-magic angle spinning) spectra, irrespective of the pH. This is due to conformational change and subsequent acquisition of intermediate time-range motions, with correlation times in the order of 10(-)(5) or 10(-)(4) s, which interferes with proton decoupling frequency or frequency of magic angle spinning, respectively, essential for an attempted peak-narrowing to achieve high-resolution NMR signals. Greater changes were achieved, however, at pH 10, which indicate large-amplitude motions of transmembrane helices upon deprotonation of Schiff base and the formation of the M-like state in the absence of illumination. The spectra detected more rapid motions in the extracellular and/or cytoplasmic loops, with correlation times increasing from 10(-)(4) to 10(-)(5) s. Conformational changes in the transmembrane helices were located at helices B, G, and D as viewed from the above-mentioned spectral changes, as well as at 1-(13)C-labeled Val 49 (helix B), 69 (B-C loop), and [3-(13)C]Ala-labeled Ala 126 (D-helix) signals, in addition to the cytoplasmic and extracellular loops. Further, we found that in the M-like state the charged state of Asp 96 at the cytoplasmic side substantially modulated the conformation and dynamics of the extracellular region through long-distance interaction.

Existence of a proton transfer chain in bacteriorhodopsin: participation of Glu-194 in the release of protons to the extracellular surface.

Glu-194 near the extracellular surface of bacteriorhodopsin is indispensable for proton release to the medium upon protonation of Asp-85 during light-driven transport. As for Glu-204, its replacement with glutamine (but not aspartate) abolishes both proton release and the anomalous titration of Asp-85 that originates from coupling between the pKa of this buried aspartate and those of the other acidic groups. Unlike the case of Glu-204, however, replacement of Glu-194 with aspartate raises the pKa for proton release. In Fourier transform infrared spectra of the E194D mutant a prominent positive band is observed at 1720 cm-1. It can be assigned from [4-13C]aspartate and D2O isotope shifts to the C&dbd;O stretch of protonated Asp-194. Its rise correlates with proton transfer from the retinal Schiff base to Asp-85. Its decay coincides with the appearance of a proton at the surface, detected under similar conditions with fluorescein covalently bound to Lys-129 and with pyranine. Its amplitude decreases with increasing pH, with a pKa of about 9. We show that this pKa is likely to be that of the internal proton donor to Asp-194, the Glu-204 site, before photoexcitation, while 13C NMR titration indicates that Asp-194 has an initial pKa of about 3. We propose that there is a chain of interacting residues between the retinal Schiff base and the extracellular surface. After photoisomerization of the retinal the pKa's change so as to allow (i) Asp-85 to become protonated by the Schiff base, (ii) the Glu-204 site to transfer its proton to Asp-194 in E194D, and therefore to Glu-194 in the wild type, and (iii) residue 194 to release the proton to the medium.

Location of a cation-binding site in the loop between helices F and G of bacteriorhodopsin as studied by 13C NMR.

The high-affinity cation-binding sites of bacteriorhodopsin (bR) were examined by solid-state 13C NMR of samples labeled with [3-13C]Ala and [1-13C]Val. We found that the 13C NMR spectra of two kinds of blue membranes, deionized (pH 4) and acid blue at pH 1.2, were very similar and different from that of the native purple membrane. This suggested that when the surface pH is lowered, either by removal of cations or by lowering the bulk pH, substantial change is induced in the secondary structure of the protein. Partial replacement of the bound cations with Na+, Ca2+, or Mn2+ produced additional spectral changes in the 13C NMR spectra. The following conclusions were made. First, there are high-affinity cation-binding sites in both the extracellular and the cytoplasmic regions, presumably near the surface, and one of the preferred cation-binding sites is located at the loop between the helix F and G (F-G loop) near Ala196, consistent with the 3D structure of bR from x-ray diffraction and cryoelectron microscopy. Second, the bound cations undergo rather rapid exchange (with a lifetime shorter than 3 ms) among various types of cation-binding sites. As expected from the location of one of the binding sites, cation binding induced conformational alteration of the F-G interhelical loop.

Long-distance effects of site-directed mutations on backbone conformation in bacteriorhodopsin from solid state NMR of [1-13C]Val-labeled proteins.

We have recorded 13C cross-polarization-magic angle spinning and dipolar decoupled-magic angle spinning NMR spectra of [1-13C]Val-labeled wild-type bacteriorhodopsin (bR), and the V49A, V199A, T46V, T46V/V49A, D96N, and D85N mutants, in order to study conformational changes of the backbone caused by site-directed mutations along the extracellular surface and the cytoplasmic half channel. On the basis of spectral changes in the V49A and V199A mutants, and upon specific cleavage by chymotrypsin, we assigned the three well-resolved 13C signals observed at 172.93, 172.00, and 171. 11 ppm to [1-13C]Val 69, Val 49, and Val 199, respectively. The local conformations of the backbone at these residues are revealed by the conformation-dependent 13C chemical shifts. We find that at the ambient temperature of these measurements Val 69 is not in a beta-sheet, in spite of previous observations by electron microscopy and x-ray diffraction at cryogenic temperatures, but in a flexible turn structure that undergoes conformational fluctuation. Results with the T46V mutant suggest that there is a long-distance effect on backbone conformation between Thr 46 and Val 49. From the spectra of the D85N and E204Q mutants there also appears to be coupling between Val 49 and Asp 85 and between Asp 85 and Glu 204, respectively. In addition, the T2 measurement indicates conformational interaction between Asp 96 and extracellular surface. The protonation of Asp 85 in the photocycle therefore might induce changes in conformation or dynamics, or both, throughout the protein, from the extracellular surface to the side chain of Asp 96.