Hideo Akutsu

Purification, characterization and reconstitution into membranes of the oligomeric c-subunit ring of thermophilic FoF1-ATP synthase expressed in Escherichia coli

F(o)F(1)-ATP synthase catalyzes ATP synthesis coupled with proton-translocation across the membrane. The membrane-embedded F(o) portion is responsible for the H(+) translocation coupled with rotation of the oligomeric c-subunit ring, which induces rotation of the γ subunit of F(1). For solid-state NMR measurements, F(o)F(1) of thermophilic Bacillus PS3 (TF(o)F(1)) was overexpressed in Escherichia coli and the intact c-subunit ring (TF(o)c-ring) was isolated by new procedures. One of the key improvement in this purification was the introduction of a His residue to each c-subunit that acts as a virtual His(10)-tag of the c-ring. After solubilization from membranes by sodium deoxycholate, the c-ring was purified by Ni-NTA affinity chromatography, followed by anion-exchange chromatography. The intactness of the isolated c-ring was confirmed by high-resolution clear native PAGE, sedimentation analysis, and H(+)-translocation activity. The isotope-labeled intact TF(o)c-ring was successfully purified in such an amount as enough for solid-state NMR measurements. The isolated TF(o)c-rings were reconstituted into lipid membranes. A solid-state NMR spectrum at a high quality was obtained with this membrane sample, revealing that this purification procedure was suitable for the investigation by solid-state NMR. The purification method developed here can also be used for other physicochemical investigations.

Active-Site Structure of the Thermophilic Foc-Subunit Ring in Membranes Elucidated by Solid-State NMR

FoF1-ATP synthase uses the electrochemical potential across membranes or ATP hydrolysis to rotate the Foc-subunit ring. To elucidate the underlying mechanism, we carried out a structural analysis focused on the active site of the thermophilic c-subunit (TFoc) ring in membranes with a solid-state NMR method developed for this purpose. We used stereo-array isotope labeling (SAIL) with a cell-free system to highlight the target. TFoc oligomers were purified using a virtual ring His tag. The membrane-reconstituted TFoc oligomer was confirmed to be a ring indistinguishable from that expressed in E.xa0coli on the basis of the H(+)-translocation activity and high-speed atomic force microscopic images. For the analysis of the active site, 2D (13)C-(13)C correlation spectra of TFoc rings labeled with SAIL-Glu and -Asn were recorded. Complete signal assignment could be performed with the aid of the C(α)i+1-C(α)i correlation spectrum of specifically (13)C,(15)N-labeled TFoc rings. The C(δ) chemical shift of Glu-56, which is essential for H(+) translocation, and related crosspeaks revealed that its carboxyl group is protonated in the membrane, forming the H(+)-locked conformation with Asn-23. The chemical shift of Asp-61 C(γ) of the E.xa0coli c ring indicated an involvement of a water molecule in the H(+) locking, in contrast to the involvement of Asn-23 in the TFoc ring, suggesting two different means of proton storage in the c rings.

Structure analysis of membrane-reconstituted subunit c-ring of E. coli H+-ATP synthase by solid-state NMR

The subunit c-ring of H+-ATP synthase (Foc-ring) plays an essential role in the proton translocation across a membrane driven by the electrochemical potential. To understand its structure and function, we have carried out solid-state NMR analysis under magic-angle sample spinning. The uniformly [13C, 15N]-labeled Foc from E. coli (EFoc) was reconstituted into lipid membranes as oligomers. Its high resolution two- and three-dimensional spectra were obtained, and the 13C and 15N signals were assigned. The obtained chemical shifts suggested that EFoc takes on a hairpin-type helix-loop-helix structure in membranes as in an organic solution. The results on the magnetization transfer between the EFoc and deuterated lipids indicated that Ile55, Ala62, Gly69 and F76 were lined up on the outer surface of the oligomer. This is in good agreement with the cross-linking results previously reported by Fillingame and his colleagues. This agreement reveals that the reconstituted EFoc oligomer takes on a ring structure similar to the intact one in vivo. On the other hand, analysis of the 13C nuclei distance of [3-13C]Ala24 and [4-13C]Asp61 in the Foc-ring did not agree with the model structures proposed for the EFoc-decamer and dodecamer. Interestingly, the carboxyl group of the essential Asp61 in the membrane-embedded EFoc-ring turned out to be protonated as COOH even at neutral pH. The hydrophobic surface of the EFoc-ring carries relatively short side chains in its central region, which may allow soft and smooth interactions with the hydrocarbon chains of lipids in the liquid-crystalline state.

Direct assignment of 13C solid-state NMR signals of TFoF1 ATP synthase subunit c-ring in lipid membranes and its implication for the ring structure

FoF1-ATP synthase catalyzes ATP hydrolysis/synthesis coupled with a transmembrane H+ translocation in membranes. The Foc-subunit ring plays a major role in this reaction. We have developed an assignment strategy for solid-state 13C NMR (ssNMR) signals of the Foc-subunit ring of thermophilic Bacillus PS3 (TFoc-ring, 72 residues), carrying one of the basic folds of membrane proteins. In a ssNMR spectrum of uniformly 13C-labeled sample, the signal overlap has been a major bottleneck because most amino acid residues are hydrophobic. To overcome signal overlapping, we developed a method designated as COmplementary Sequential assignment with MInimum Labeling Ensemble (COSMILE). According to this method, we generated three kinds of reverse-labeled samples to suppress signal overlapping. To assign the carbon signals sequentially, two-dimensional Cα(i+1)–C′Cα(i) correlation and dipolar assisted rotational resonance (DARR) experiments were performed under magic-angle sample spinning. On the basis of inter- and intra-residue 13C–13C chemical shift correlations, 97% of Cα, 97% of Cβ and 92% of C′ signals were assigned directly from the spectra. Secondary structure analysis predicted a hairpin fold of two helices with a central loop. The effects of saturated and unsaturated phosphatidylcholines on TFoc-ring structure were examined. The DARR spectra at 15xa0ms mixing time are essentially similar to each other in saturated and unsaturated lipid membranes, suggesting that TFoc-rings have similar structures under the different environments. The spectrum of the sample in saturated lipid membranes showed better resolution and structural stability in the gel state. The C-terminal helix was suggested to locate in the outer layer of the c-ring.

CHAPTER 3:13C–13C Distance Measurements by Polarization Transfer Matrix Analysis of 13C Spin Diffusion in a Uniformly 13C-Labeled Molecular Complex under Magic Angle Spinning

Spin diffusion of 13C polarization in NMR provides 13C−13C distances under magic angle spinning over a broad spectral width. However, there are difficulties in obtaining the distances accurately in uniformly 13C-labeled molecular complexes in solids. Effects of the weak long-range couplings are suppressed by strong short-range couplings. In addition, direct polarization transfer should be distinguished from relayed transfer. To address these issues, polarization-transfer rate matrix analysis has been applied to the 13C-driven spin diffusion in a uniformly 13C-labeled bacteriochlorophyll c assembly. The transfer rates due to direct dipolar couplings were derived by matrix analysis. Distances were obtained from the rates by perturbation theory for spin diffusion using zero-quantum lineshapes. This procedure gave distances up to 6 A with an accuracy of 25−50%. Correction of the distances from the zero-quantum lineshapes improved the accuracy by about 5−15%. These results show that rate matrix analysis is beneficial for distance analysis of molecular complexes for solid-state NMR. Also, the coefficient and anisotropy of 13C spin diffusion in solids are discussed quantitatively.