Hazime Saitô

Surface and dynamic structures of bacteriorhodopsin in a 2D crystal, a distorted or disrupted lattice, as revealed by site-directed solid-state 13C NMR

The 3D structure of bacteriorhodopsin (bR) obtained by X‐ray diffraction or cryo‐electron microscope studies is not always sufficient for a picture at ambient temperature where dynamic behavior is exhibited. For this reason, a site‐directed solid‐state 13C NMR study of fully hydrated bR from purple membrane (PM), or a distorted or disrupted lattice, is very valuable in order to gain insight into the dynamic picture. This includes the surface structure, at the physiologically important ambient temperature. Almost all of the 13C NMR signals are available from [3‐13C]Ala or [1‐13C]Val‐labeled bR from PM, although the 13C NMR signals from the surface areas, including loops and transmembrane α‐helices near the surface (8.7u2003Å depth), are suppressed for preparations labeled with [1‐13C]Gly, Ala, Leu, Phe, Tyr, etc. due to a failure of the attempted peak‐narrowing by making use of the interfered frequency of the frequency of fluctuation motions with the frequency of magic angle spinning. In particular, the C‐terminal residues, 226–235, are present as the C‐terminal α‐helix which is held together with the nearby loops to form a surface complex, although the remaining C‐terminal residues undergo isotropic motion even in a 2D crystalline lattice (PM) under physiological conditions. Surprisingly, the 13C NMR signals could be further suppressed even from [3‐13C]Ala‐ or [1‐13C]Val‐bR, due to the acquired fluctuation motions with correlation times in the order of 10−4 to 10−5u2003s, when the 2D lattice structure is instantaneously distorted or completely disrupted, either in photo‐intermediate, removed retinal or when embedded in the lipid bilayers.

Pressure induced isomerization of retinal on bacteriorhodopsin as disclosed by Fast Magic Angle Spinning NMR

Bacteriorhodopsin (bR) is a retinal protein in purple membrane of Halobacterium salinarum, which functions as a light‐driven proton pump. We have detected pressure‐induced isomerization of retinal in bR by analyzing 15N cross polarization‐magic angle spinning (CP‐MAS) NMR spectra of [ζ‐15N]Lys‐labeled bR. In the 15N‐NMR spectra, both all‐trans and 13‐cis retinal configurations have been observed in the Lys Nζ in protonated Schiff base at 148.0 and 155.0u2003ppm, respectively, at the MAS frequency of 4u2003kHz in the dark. When the MAS frequency was increased up to 12u2003kHz corresponding to the sample pressure of 63 bar, the 15N‐NMR signals of [ζ‐15N]Lys in Schiff base of retinal were broadened. On the other hand, other [ζ‐15N]Lys did not show broadening. Subsequently, the increased signal intensity of [ζ‐15N]Lys in Schiff base of 13‐cis retinal at 155.0u2003ppm was observed when the MAS frequency was decreased from 12 to 4u2003kHz. These results showed that the equilibrium constant of [all‐trans‐bR]/[13‐cis‐bR] in retinal decreased by the pressure of 63 bar. It was also revealed that the structural changes induced by the pressure occurred in the vicinity of retinal. Therefore, microscopically, hydrogen‐bond network around retinal would be disrupted or distorted by a constantly applied pressure. It is, therefore, clearly demonstrated that increased pressure induced by fast MAS frequencies generated isomerization of retinal from all‐trans to 13‐cis state in the membrane protein bR.

Dynamics Change of Phoborhodopsin and Transducer by Activation: Study Using D75N Mutant of the Receptor by Site-directed Solid-state 13C NMR†

Pharaonis phoborhodopsin (ppR or sensory rhodopsin II) is a negative phototaxis receptor of Natronomonas pharaonis, and forms a complex, which transmits the photosignal into cytoplasm, with its cognate transducer (pHtrII). We examined a possible local dynamics change of ppR and its D75N mutant complexed with pHtrII, using solid‐state 13C NMR of [3‐13C]Ala‐ and [1‐13C]Val‐labeled preparations. We distinguished Ala Cβ13C signals of relatively static stem (Ala221) in the C‐terminus of the receptors from those of flexible tip (Ala228, 234, 236 and 238), utilizing a mutant with truncated C‐terminus. The local fluctuation frequency at the C‐terminal tip was appreciably decreased when ppR was bound to pHtrII, while it was increased when D75N, that mimics the signaling state because of disrupted salt bridge between C and G helices prerequisite for the signal transfer, was bound to pHtrII. This signal change may be considered with the larger dissociation constant of the complex between pHtrII and M‐state of ppR. At the same time, it turned out that fluctuation frequency of cytoplasmic portion of pHtrII is lowered when ppR is replaced by D75N in the complex with pHtrII. This means that the C‐terminal tip partly participates in binding with the linker region of pHtrII in the dark, but this portion might be released at the signaling state leading to mutual association of the two transducers in the cytoplasmic regions within the ppR/pHtrII complex.

Participation of the surface structure of pharaonis phoborhodopsin, ppR and its A149S and A149V mutants, consisting of the C-terminal α-helix and E-F loop, in the complex-formation with the cognate transducer pHtrII, as revealed by site-directed 13C solid-state NMR

We have recorded 13C solid state NMR spectra of [3‐13C]Ala‐labeled pharaonis phoborhodopsin (ppR) and its mutants, A149S and A149V, complexed with the cognate transducer pharaonis halobacterial transducer II protein (pHtrII) (1–159), to gain insight into a possible role of their cytoplasmic surface structure including the C‐terminal α‐helix and E–F loop for stabilization of the 2:2 complex, by both cross‐polarization magic angle spinning (CP‐MAS) and dipolar decoupled (DD)‐MAS NMR techniques. We found that 13C CP‐MAS NMR spectra of [3‐13C]Ala‐ppR, A149S and A149V complexed with the transducer pHtrII are very similar, reflecting their conformation and dynamics changes caused by mutual interactions through the transmembrane α‐helical surfaces. In contrast, their DD‐MAS NMR spectral features are quite different between [3‐13C]Ala‐ A149S and A149V in the complexes with pHtrII: 13C DD‐MAS NMR spectrum of [3‐13C]Ala‐A149S complex is rather similar to that of the uncomplexed form, while the corresponding spectral feature of A149V complex is similar to that of ppR complex in the C‐terminal tip region. This is because more flexible surface structure detected by the DD‐MAS NMR spectra are more directly influenced by the dynamics changes than the CP‐MAS NMR. It turned out, therefore, that an altered surface structure of A149S resulted in destabilized complex as viewed from the 13C NMR spectrum of the surface areas, probably because of modified conformation at the corner of the helix E in addition to the change of hydropathy. It is, therefore, concluded that the surface structure of ppR including the C‐terminal α‐helix and the E–F loops is directly involved in the stabilization of the complex through conformational stability of the helix E.

Site-Directed Solid-State NMR on Membrane Proteins

Well-resolved, fully visible 13 C NMR signals of membrane proteins were successfully recorded either at ambient or at lower temperatures when they were embedded in lipid bilayers constituting a 2D crystalline lattice, as manifested from site-directed 13 C NMR studies on [3- 13 C]Ala and/or [1- 13 C]Val-labeled bacteriorhodopsin (bR) from purple membrane, instead of uniformly labeled preparations. It is emphasized that recording 13 C NMR spectra by dipolar decoupled magic angle spinning (DD-MAS) is essential to detect signals from rather flexible portions of fully hydrated membrane proteins, in addition to recording signals from static portions by cross-polarization-magic angle spinning (CP-MAS). The resulting 13 C NMR signals were site-directly assigned to specific residues utilizing site-directed mutants, otherwise global conformational changes were introduced by such mutations. Conformational features of bR with emphasis on surface area, as defined by surface complex, were discussed together with its biological significance, on the basis of the conformation-dependent displacement of the 13 C chemical shifts. Further, slow local membrane dynamics with frequencies of 10 5 or 10 4 xa0Hz, which is important for biological functions, was analyzed as viewed from specifically suppressed 13 C NMR signals of certain regions caused by interference of fluctuation frequency with frequency of proton-decoupling or magic angle spinning. This approach has been further extended to reveal the conformation and dynamics of a variety of 13 C-labeled membrane proteins that are overexpressed from Escherichia coli and subsequently not always involved in a 2D crystalline lattice by 13 C NMR: they include phototaxis receptor protein (pharaonis phoborhodopsin), its transducer (pHtrII), and membrane enzyme (diacylglycerol kinase). In such cases, it was found that several 13 C NMR signals could be suppressed from amino-acid residues located at the flexible portions such as loops and transmembrane α-helices near to the membrane surface, as a result of interference of dominant frequency for conformational fluctuations of the order of 10 4 -10 5 xa0Hz. This means that protein–protein contact in the 2D crystalline lattice significantly regulates the protein dynamics. It is therefore important to search for the most appropriate experimental condition to be able to observe the full 13 C NMR signals of the membrane proteins under consideration, including the choice of the most appropriate 13 C-labeled amino acids, temperature, ionic state, etc.

Suppressed or recovered intensities analysis in site-directed 13C NMR: Assessment of low-frequency fluctuations in bacteriorhodopsin and D85N mutants revisited

The first proton transfer of bacteriorhodopsin (bR) occurs from the protonated Schiff base to the anionic Asp 85 at the central part of the protein in the L to M states. Low-frequency dynamics accompanied by this process can be revealed by suppressed or recovered intensities (SRI) analysis of site-directed (13)C solid-state NMR spectra of 2D crystalline preparations. First of all, we examined a relationship of fluctuation frequencies available from [1-(13)C]Val- and [3-(13)C]Ala-labeled preparations, by taking the effective correlation time of internal methyl rotations into account. We analyzed the SRI data of [1-(13)C]Val-labeled wild-type bR and D85N mutants, as a function of temperature and pH, respectively, based on so-far assigned peaks including newly assigned or revised ones. Global conformational change of the protein backbone, caused by neutralization of the anionic D85 by D85N, can be visualized by characteristic displacement of peaks due to the conformation-dependent (13)C chemical shifts. Concomitant dynamics changes if any, with fluctuation frequencies in the order of 10(4) Hz, were evaluated by the decreased peak intensities in the B-C and D-E loops of D85N mutant. The resulting fluctuation frequencies, owing to subsequent, accelerated dynamics changes in the M-like state by deprotonation of the Schiff base at alkaline pH, were successfully evaluated based on the SRI plots as a function of pH, which were varied depending upon the extent of interference of induced fluctuation frequency with frequency of magic angle spinning or escape from such interference. Distinguishing fluctuation frequencies between the higher and lower than 10(4) Hz is now possible, instead of a simple description of the data around 10(4) Hz available from one-point data analysis previously reported.

Natural abundance 13C REDOR coupled to a singly 15N-labeled nucleus: simultaneous determination of interatomic distances in crystalline ammonium [15N] l-glutamate monohydrate

Abstract REDOR technique was applied to natural abundance 13 C nuclei coupled to a singly labeled 15 N nucleus to determine the 13 C, 15 N interatomic distances simultaneously in crystalline ammonium [ 15 N] l -glutamate monohydrate ( 1 ). Consequently, the interatomic C–N distances between 15 N and 13 C O, 13 C α , 13 C β , 13 C γ , and 13 C δ carbon nuclei for 1 were determined with a precision of ±0.15xa0A, after the experimental conditions such as the location of samples in the rotor, length of π pulse etc. were carefully optimized. 13 C-REDOR factors for three spin system, (Δ S / S 0 ) CN1N2 , and the sum of two isolated 2-spin system, ( Δ S/S 0 ) ∗ =( Δ S/S 0 ) CN 1 +( Δ S/S 0 ) CN 2 , were further evaluated by the REDOR measurements on isotopically diluted 1 in a controlled manner. Subsequently, the intra- and intermolecular C–N distances were separated by searching the minima in the contour map of root mean square deviation (RMSD) between the theoretically and experimentally obtained ( Δ S/S 0 ) ∗ values against two interatomic distances, r C–N1 and r C–N2 . When the intramolecular C–N distance ( r C–N1 ) of the particular carbon nucleus is substantially shorter than the intermolecular one ( r C–N2 ), C–N distances within a single molecule were obtained with an accuracy of ±0.06xa0A as in the cases of C O, C α and C β carbon nuclei. C–N distances between the molecule in question and the nearest neighboring molecules can be also obtained, although accuracy was lower. On the contrary, it was difficult to determine the interatomic distances in the same molecule when the intermolecular dipolar contribution is larger than the intramolecular one as in the case of C δ carbon nucleus.

Site-Directed NMR Studies on Membrane Proteins

Integral membrane proteins, traversing the membrane once or several times as α-helices, play crucial roles in maintaining various activities of cells such as transport of appropriate molecules into or out of the cell, catalysis of chemical reaction, and receiving and transducing chemical signals from the cell environment. Naturally, biological activity of such proteins may depend upon their conformations and dynamics regulated by specific lipid– protein and/or protein–protein interactions as structural determinants, as studied by analysis of 2D assembly of bacteriorhodopsin (bR) as a typical membrane protein [1]. bR is active as a proton pump and considered as a prototype of a variety of G-protein coupled receptors, consisting of seven transmembrane α-helices. Interestingly, the bR structure is far from static at ambient temperature in spite of currently available 3D structural models revealed by crystallography at low temperature but flexible even in the 2D crystal, especially at the loops and Nor C-terminal residues fully exposed to aqueous phase, and undergoing fluctuation motions with correlation times of the order of 10−4–10−5 and 10−8 s, respectively, as revealed by recent site-directed solid-state 13C NMR[2–6]. Well-resolved 13C NMR signals are fully visible from 2D crystalline 13C-labeled [3-13C]Ala-[2,3,7] or [1-13C]Val-labeled bR[3,5] recorded by both crosspolarization-magic angle spinning (CP-MAS) and dipolar decoupled-magic angle spinning (DD-MAS) techniques. Inherent motional fluctuation of the transmembrane αhelices of bR monomer, however, turns out to be accelerated by two orders of magnitude in the lipid bilayer in the absence of specific protein–protein interactions, from their correlation times of the order of 10−2 s in 2D crystal [2,3,8] to 10−4–10−5 s, [9–13] in the monomer. Accordingly, 13C NMR signals from several residues in the transmembrane α-helices and loops could be suppressed due to the failure of attempted peak-narrowing caused by interference of the motional fluctuation frequency with the frequency of proton decoupling or MAS [2,3,14–16], although the functional unit responsible for the photocycle is the monomer itself rather than the trimeric form found in the 2D crystal [17,18]. In this case, uniform 13C-labeling is not always favorable for solid-state NMR, because 13C NMR study of densely 13C-labeled proteins such as [1,2,3-C3]Ala-labeled bR could be substantially broadened in the presence of such intermediate and slow motions, due to the accelerated relaxation rate through a number of homonuclear 13C–13C dipolar interactions and scalar J couplings [16]. We demonstrate here how the site-directed 13C NMR approach is useful to reveal conformational features of intact membrane proteins with emphasis on their surface structures and dynamics at ambient temperature, as revealed by 13C NMR studies on bR from the 2D crystal and monomer and various membrane proteins active as signal transducers and enzyme, expressed from E. coli and present as monomer in lipid bilayers.